Structured Review

Becton Dickinson rat monoclonal anti mouse syndecan 4
ATXβ controls breast cancer cell metastasis through an SDC4-dependent mechanism ( A ) 4T1 cell adhesion to increasing amounts of ATXβ, BSA (400 ng) was used as control (left panels). Data represent the mean of adherent cells/mm 2 ±SD of adherent cells of 3 experiments performed in 8 replicates (right panel). ( B ) Flow cytometry detection of cell surface expression of <t>syndecan-4</t> (SDC4) in 4T1 cells. Cells were immunostained with KY/8.2 monoclonal antibody (anti-SDC4) (black bar), or isotype control antibody MOPC21 (grey bar). NT: not treated cells (open bar). ( C ) Inhibition of 4T1 cell adhesion on ATXβ with KY/8.2 antibody (anti-SDC4). Indicated cell lines were preincubated for 1 h in the presence of KY/8.2 or MOPC21 antibodies (10 µg/mL). Data represent the mean of adherent cells/mm 2 ±SD of adherent cells of 3 experiments performed in 8 replicates ( *** : P
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Images

1) Product Images from "Autotaxin-β interaction with the cell surface via syndecan-4 impacts on cancer cell proliferation and metastasis"

Article Title: Autotaxin-β interaction with the cell surface via syndecan-4 impacts on cancer cell proliferation and metastasis

Journal: Oncotarget

doi: 10.18632/oncotarget.26039

ATXβ controls breast cancer cell metastasis through an SDC4-dependent mechanism ( A ) 4T1 cell adhesion to increasing amounts of ATXβ, BSA (400 ng) was used as control (left panels). Data represent the mean of adherent cells/mm 2 ±SD of adherent cells of 3 experiments performed in 8 replicates (right panel). ( B ) Flow cytometry detection of cell surface expression of syndecan-4 (SDC4) in 4T1 cells. Cells were immunostained with KY/8.2 monoclonal antibody (anti-SDC4) (black bar), or isotype control antibody MOPC21 (grey bar). NT: not treated cells (open bar). ( C ) Inhibition of 4T1 cell adhesion on ATXβ with KY/8.2 antibody (anti-SDC4). Indicated cell lines were preincubated for 1 h in the presence of KY/8.2 or MOPC21 antibodies (10 µg/mL). Data represent the mean of adherent cells/mm 2 ±SD of adherent cells of 3 experiments performed in 8 replicates ( *** : P
Figure Legend Snippet: ATXβ controls breast cancer cell metastasis through an SDC4-dependent mechanism ( A ) 4T1 cell adhesion to increasing amounts of ATXβ, BSA (400 ng) was used as control (left panels). Data represent the mean of adherent cells/mm 2 ±SD of adherent cells of 3 experiments performed in 8 replicates (right panel). ( B ) Flow cytometry detection of cell surface expression of syndecan-4 (SDC4) in 4T1 cells. Cells were immunostained with KY/8.2 monoclonal antibody (anti-SDC4) (black bar), or isotype control antibody MOPC21 (grey bar). NT: not treated cells (open bar). ( C ) Inhibition of 4T1 cell adhesion on ATXβ with KY/8.2 antibody (anti-SDC4). Indicated cell lines were preincubated for 1 h in the presence of KY/8.2 or MOPC21 antibodies (10 µg/mL). Data represent the mean of adherent cells/mm 2 ±SD of adherent cells of 3 experiments performed in 8 replicates ( *** : P

Techniques Used: Flow Cytometry, Cytometry, Expressing, Inhibition

2) Product Images from "STIM1 deficiency is linked to Alzheimer’s disease and triggers cell death in SH-SY5Y cells by upregulation of L-type voltage-operated Ca2+ entry"

Article Title: STIM1 deficiency is linked to Alzheimer’s disease and triggers cell death in SH-SY5Y cells by upregulation of L-type voltage-operated Ca2+ entry

Journal: Journal of Molecular Medicine (Berlin, Germany)

doi: 10.1007/s00109-018-1677-y

Loss of cell viability in differentiating STIM1-KO cells. a Cell viability of wild-type (black bars) and STIM1-KO cells (gray bars) was evaluated with an MTT assay at different stages of differentiation: undifferentiated cells, 24 h in growing medium (1 DIV); 24 h in growing medium + 2 days in differentiating medium (3 DIV); 24 h in growing medium + 5 days in differentiating medium (6 DIV). Data are presented as mean ± s.d. of three independent experiments, and results are normalized to the values obtained from wild-type cells at 6 DIV. b The analysis of the cell cycle was performed in undifferentiated (1 DIV) and differentiated cells (6 days of differentiation), and the assay was performed by staining fixed cells with propidium iodide and analyzing cells by flow cytometry. The percentage of cells at G2/M phase is plotted in the right panel to show the statistically significant increase of this phase in STIM1-KO differentiated cells. In both panels, data are the mean ± s.d. of three independent experiments. c Cells were cultured as indicated above, and stained with C12FDG to evaluate senescence by flow cytometry. The top panels show representative data histograms, with data from unstained cells as negative control (in orange), undifferentiated cells (pink), and differentiated cells after 6 DIV (blue). The y -axis is the normalized cell number, and the x -axis is the fluorescence intensity from C12FDG. Data of four independent experiments are shown in the bottom panel as mean ± s.d. d Lysates from cells in the experimental conditions described for panels ( b , c ) were assessed for p21 expression by immunoblot. The top panel shows a representative blot, with GAPDH as a loading control. The bottom panel shows data of four independent experiments as mean ± s.d.
Figure Legend Snippet: Loss of cell viability in differentiating STIM1-KO cells. a Cell viability of wild-type (black bars) and STIM1-KO cells (gray bars) was evaluated with an MTT assay at different stages of differentiation: undifferentiated cells, 24 h in growing medium (1 DIV); 24 h in growing medium + 2 days in differentiating medium (3 DIV); 24 h in growing medium + 5 days in differentiating medium (6 DIV). Data are presented as mean ± s.d. of three independent experiments, and results are normalized to the values obtained from wild-type cells at 6 DIV. b The analysis of the cell cycle was performed in undifferentiated (1 DIV) and differentiated cells (6 days of differentiation), and the assay was performed by staining fixed cells with propidium iodide and analyzing cells by flow cytometry. The percentage of cells at G2/M phase is plotted in the right panel to show the statistically significant increase of this phase in STIM1-KO differentiated cells. In both panels, data are the mean ± s.d. of three independent experiments. c Cells were cultured as indicated above, and stained with C12FDG to evaluate senescence by flow cytometry. The top panels show representative data histograms, with data from unstained cells as negative control (in orange), undifferentiated cells (pink), and differentiated cells after 6 DIV (blue). The y -axis is the normalized cell number, and the x -axis is the fluorescence intensity from C12FDG. Data of four independent experiments are shown in the bottom panel as mean ± s.d. d Lysates from cells in the experimental conditions described for panels ( b , c ) were assessed for p21 expression by immunoblot. The top panel shows a representative blot, with GAPDH as a loading control. The bottom panel shows data of four independent experiments as mean ± s.d.

Techniques Used: MTT Assay, Staining, Flow Cytometry, Cytometry, Cell Culture, Negative Control, Fluorescence, Expressing

Loss of mitochondrial function in STIM1-KO differentiated cells. a Cells, differentiated as indicated in Fig. 2 , were stained with rhodamine 123 in phenol red and serum-free medium, and observed under epifluorescence. Samples, in bicarbonate-free Leibovitz’s L-15 medium, were live-cell imaged at 37 °C in an UNO-Okolab stage incubator and analyzed with a Plan Apochromat × 100 (NA 1.45) oil immersion objective. Z-sections (0.2-μm steps) were recorded, and after deconvolution, the total projection was analyzed to observe mitochondria morphology. Representative images from two independent experiments are shown ( > 20 cells per condition). Dashed line depicts nuclear envelope. Scale bar = 10 μm. b Cells, under the experimental conditions described for panel ( a ), were stained with TMRM, and the fraction of positively TMRM-stained cells was analyzed by flow cytometry. Data are presented as mean ± s.d. of three independent experiments. c Cells were cultured on glass coverslips and stained with TMRM. Cells were visualized under confocal microscopy to evaluate the intensity and morphology of staining in resting conditions. FCCP (10 μM) was added to assess non-specific staining and loss of signal due to mitochondrial inner membrane depolarization after 2 and 4 min. Hoechst 33342 was used to stain cell nuclei (blue). Scale bar = 10 or 20 μm, as indicated. TMRM fluorescence intensity from different ROIs was quantified, and the data (mean ± s.d. of two independent experiments) are shown as a box plot (right panel). d TMRM-stained cells were analyzed by flow cytometry. Histograms (left panel) depict the number of events ( y -axis) and fluorescence intensity ( x -axis) for wild-type and STIM1-KO cells before and 10-min after addition of 10 μM FCCP. Total fluorescence data of two independent experiments are shown in the right panel bar chart as mean ± s.d.
Figure Legend Snippet: Loss of mitochondrial function in STIM1-KO differentiated cells. a Cells, differentiated as indicated in Fig. 2 , were stained with rhodamine 123 in phenol red and serum-free medium, and observed under epifluorescence. Samples, in bicarbonate-free Leibovitz’s L-15 medium, were live-cell imaged at 37 °C in an UNO-Okolab stage incubator and analyzed with a Plan Apochromat × 100 (NA 1.45) oil immersion objective. Z-sections (0.2-μm steps) were recorded, and after deconvolution, the total projection was analyzed to observe mitochondria morphology. Representative images from two independent experiments are shown ( > 20 cells per condition). Dashed line depicts nuclear envelope. Scale bar = 10 μm. b Cells, under the experimental conditions described for panel ( a ), were stained with TMRM, and the fraction of positively TMRM-stained cells was analyzed by flow cytometry. Data are presented as mean ± s.d. of three independent experiments. c Cells were cultured on glass coverslips and stained with TMRM. Cells were visualized under confocal microscopy to evaluate the intensity and morphology of staining in resting conditions. FCCP (10 μM) was added to assess non-specific staining and loss of signal due to mitochondrial inner membrane depolarization after 2 and 4 min. Hoechst 33342 was used to stain cell nuclei (blue). Scale bar = 10 or 20 μm, as indicated. TMRM fluorescence intensity from different ROIs was quantified, and the data (mean ± s.d. of two independent experiments) are shown as a box plot (right panel). d TMRM-stained cells were analyzed by flow cytometry. Histograms (left panel) depict the number of events ( y -axis) and fluorescence intensity ( x -axis) for wild-type and STIM1-KO cells before and 10-min after addition of 10 μM FCCP. Total fluorescence data of two independent experiments are shown in the right panel bar chart as mean ± s.d.

Techniques Used: Staining, Flow Cytometry, Cytometry, Cell Culture, Confocal Microscopy, Fluorescence

Increased cellular Ca 2+ influx underlies mitochondrial failure and augmented senescence. a Changes in cytosolic-free Ca 2+ concentration were analyzed in fura-2-loaded cells. Cells in HBSS containing 1.26 mM Ca 2+ were subjected to 1-min depolarization with 90 mM KCl (red line). CaCl 2 in the HBSS was increased to 5 mM during depolarization to facilitate the Ca 2+ influx recording. In parallel experiments, 10 μM nifedipine was added to the assay medium during the recording (black line). Right panel: the increase of the F340/F380 ratio triggered by 90 mM KCl in the presence of VOCCs blockers is shown as mean ± s.d. of 3 experiments (a minimum of 70 cells per experimental condition). Final concentrations: 10 μM nifedipine, 1 μM ω-conotoxin MVIIC, 3 μM ML 218. b STIM1-KO cells, or STIM1-KO cells stably expressing a specific shRNA to knock-down CACNA1C transcripts, were treated as described in panel ( a ). The left panel shows a representative experiment, and the bar chart of the right panel shows the increase in the F340/F380 ratio evoked by depolarization (mean ± s.d. of two independent experiments; n > 60 cells per condition). c Senescence (left panel) and mitochondrial polarization (middle and right panels) were assessed from differentiated cells after 6 DIV, staining with C12FDG as described in Fig. 5 c and TMRM as in Fig. 6 b–d, respectively. Data are mean ± s.d. of three independent experiments (number of replicates is shown for each condition). d Rotenone-sensitive NADH oxidase activity was assessed from differentiated SH-SY5Y cell lysates (wild-type, STIM1-KO, and STIM1-KO + shRNA for CACNA1C ). Data are presented as the mean ± s.d. of two independent experiments. e Cell were transiently transfected for the expression of the Ca 2+ sensor 4mtD3cpv. Mitochondrial [Ca 2+ ] was assessed as described in Fig. 7 . Data of six independent experiments are shown in the right panel bar chart as mean ± s.d.
Figure Legend Snippet: Increased cellular Ca 2+ influx underlies mitochondrial failure and augmented senescence. a Changes in cytosolic-free Ca 2+ concentration were analyzed in fura-2-loaded cells. Cells in HBSS containing 1.26 mM Ca 2+ were subjected to 1-min depolarization with 90 mM KCl (red line). CaCl 2 in the HBSS was increased to 5 mM during depolarization to facilitate the Ca 2+ influx recording. In parallel experiments, 10 μM nifedipine was added to the assay medium during the recording (black line). Right panel: the increase of the F340/F380 ratio triggered by 90 mM KCl in the presence of VOCCs blockers is shown as mean ± s.d. of 3 experiments (a minimum of 70 cells per experimental condition). Final concentrations: 10 μM nifedipine, 1 μM ω-conotoxin MVIIC, 3 μM ML 218. b STIM1-KO cells, or STIM1-KO cells stably expressing a specific shRNA to knock-down CACNA1C transcripts, were treated as described in panel ( a ). The left panel shows a representative experiment, and the bar chart of the right panel shows the increase in the F340/F380 ratio evoked by depolarization (mean ± s.d. of two independent experiments; n > 60 cells per condition). c Senescence (left panel) and mitochondrial polarization (middle and right panels) were assessed from differentiated cells after 6 DIV, staining with C12FDG as described in Fig. 5 c and TMRM as in Fig. 6 b–d, respectively. Data are mean ± s.d. of three independent experiments (number of replicates is shown for each condition). d Rotenone-sensitive NADH oxidase activity was assessed from differentiated SH-SY5Y cell lysates (wild-type, STIM1-KO, and STIM1-KO + shRNA for CACNA1C ). Data are presented as the mean ± s.d. of two independent experiments. e Cell were transiently transfected for the expression of the Ca 2+ sensor 4mtD3cpv. Mitochondrial [Ca 2+ ] was assessed as described in Fig. 7 . Data of six independent experiments are shown in the right panel bar chart as mean ± s.d.

Techniques Used: Concentration Assay, Stable Transfection, Expressing, shRNA, Staining, Activity Assay, Transfection

Expression levels of STIM1 in human tissue samples. a Samples of medium frontal gyrus membranes (12 μg) from human tissues characterized for diagnosis as non-AD control Braak stage I (first lane) and AD-Braak stages IV, V, and VI, were electrophoresed in a 7.5% SDS-PAGE gel, electrotransferred to nitrocellulose and immunostained with anti-STIM1 antibody. A representative immunoblot from four assays is shown. b Quantification of STIM1 protein level relative to β-tubulin is shown as mean ± SE values (a.u., arbitrary units)
Figure Legend Snippet: Expression levels of STIM1 in human tissue samples. a Samples of medium frontal gyrus membranes (12 μg) from human tissues characterized for diagnosis as non-AD control Braak stage I (first lane) and AD-Braak stages IV, V, and VI, were electrophoresed in a 7.5% SDS-PAGE gel, electrotransferred to nitrocellulose and immunostained with anti-STIM1 antibody. A representative immunoblot from four assays is shown. b Quantification of STIM1 protein level relative to β-tubulin is shown as mean ± SE values (a.u., arbitrary units)

Techniques Used: Expressing, SDS Page

STIM1 expression during differentiation of SH-SY5Y cells. a Top: SH-SY5Y cells were differentiated with RA + BDNF, and bright-field microscopy images of cells were recorded (left panel, non-differentiated; right panel, differentiated after 9 DIV). Bottom: Neurite length was measured in undifferentiated cells ( n = 52), and differentiated cells ( n = 45), from two independent cultures. Scale bar = 100 μm. b Top: Expression of STIM1 and TUBB3 was assessed by immunoblot from undifferentiated cells and cells differentiated after 9–10 DIV with RA + BDNF. Level of GAPDH was assessed as a loading control of the immunoblot. Bottom: The expression of STIM1 and TUBB3 was quantified by immunoblotting with lysates from three independent assays. c Store-operated Ca 2+ entry was evaluated in undifferentiated (black line) and differentiated cells (red line). Fura-2-loaded cells were incubated in a Ca 2+ -free HBSS (assay medium), and 1 μM thapsigargin (Tg) was added to the cells for 6 min. Ca 2+ (2 mM CaCl 2 ) was finally added to the cells to evaluate the extension of Ca 2+ -entry. The experiment was performed at controlled temperature (36–37 °C). Data are presented as the mean ± s.d. of three independent experiments ( n > 60 cells for each condition)
Figure Legend Snippet: STIM1 expression during differentiation of SH-SY5Y cells. a Top: SH-SY5Y cells were differentiated with RA + BDNF, and bright-field microscopy images of cells were recorded (left panel, non-differentiated; right panel, differentiated after 9 DIV). Bottom: Neurite length was measured in undifferentiated cells ( n = 52), and differentiated cells ( n = 45), from two independent cultures. Scale bar = 100 μm. b Top: Expression of STIM1 and TUBB3 was assessed by immunoblot from undifferentiated cells and cells differentiated after 9–10 DIV with RA + BDNF. Level of GAPDH was assessed as a loading control of the immunoblot. Bottom: The expression of STIM1 and TUBB3 was quantified by immunoblotting with lysates from three independent assays. c Store-operated Ca 2+ entry was evaluated in undifferentiated (black line) and differentiated cells (red line). Fura-2-loaded cells were incubated in a Ca 2+ -free HBSS (assay medium), and 1 μM thapsigargin (Tg) was added to the cells for 6 min. Ca 2+ (2 mM CaCl 2 ) was finally added to the cells to evaluate the extension of Ca 2+ -entry. The experiment was performed at controlled temperature (36–37 °C). Data are presented as the mean ± s.d. of three independent experiments ( n > 60 cells for each condition)

Techniques Used: Expressing, Microscopy, Incubation

STIM1 deficiency did not modify markers of differentiation. a STIM1-KO cells and the parental cell line were differentiated as indicated above, and images of cells in culture were recorded to assess neurite length in undifferentiated cells (top panels), and differentiated after 12 DIV of treatment (bottom panels). Scale bar = 200 μm. Quantification of neurite length revealed no differences between wild-type and STIM1-KO cells. Data are presented as the mean ± s.d. of two independent experiments ( n = 50 cells for KO; n = 45 cells for wt). b TUBB3 expression was studied by immunoblot, as in Fig. 2 , using GAPDH as a loading control. Differentiation was stopped at 9 DIV and the relative expression of TUBB3 was assessed in three independent experiments (data are the mean ± s.d.)
Figure Legend Snippet: STIM1 deficiency did not modify markers of differentiation. a STIM1-KO cells and the parental cell line were differentiated as indicated above, and images of cells in culture were recorded to assess neurite length in undifferentiated cells (top panels), and differentiated after 12 DIV of treatment (bottom panels). Scale bar = 200 μm. Quantification of neurite length revealed no differences between wild-type and STIM1-KO cells. Data are presented as the mean ± s.d. of two independent experiments ( n = 50 cells for KO; n = 45 cells for wt). b TUBB3 expression was studied by immunoblot, as in Fig. 2 , using GAPDH as a loading control. Differentiation was stopped at 9 DIV and the relative expression of TUBB3 was assessed in three independent experiments (data are the mean ± s.d.)

Techniques Used: Expressing

Mitochondrial electron transport complex and mitochondrial Ca 2+ levels. a Total NADH oxidase activity and rotenone-sensitive activity was assessed from differentiated SH-SY5Y cell lysates (WT and STIM1-KO). Data are presented as the mean ± s.d. of two independent experiments. Right panel shows the difference between total activity and the remaining activity after rotenone addition to the assay, i.e., the rotenone-sensitive NADH oxidase. b Wild-type and STIM1-KO cells were transiently transfected for the expression of the Ca 2+ sensor 4mtD3cpv and 48 h later emission of fluorescence was recorded for CFP, FRET (left and middle panels,), and YFP channels to monitor photobleaching. FRET/CFP ratio signal (right panel) was recorded for cells in Ca 2+ -containing HBSS for 4–5 min. Calibration of FRET/CFP ratio to calculate Rmin and Rmax was performed individually for every assay. [Ca 2+ ] m data are presented as the mean ± s.d. of seven independent experiments
Figure Legend Snippet: Mitochondrial electron transport complex and mitochondrial Ca 2+ levels. a Total NADH oxidase activity and rotenone-sensitive activity was assessed from differentiated SH-SY5Y cell lysates (WT and STIM1-KO). Data are presented as the mean ± s.d. of two independent experiments. Right panel shows the difference between total activity and the remaining activity after rotenone addition to the assay, i.e., the rotenone-sensitive NADH oxidase. b Wild-type and STIM1-KO cells were transiently transfected for the expression of the Ca 2+ sensor 4mtD3cpv and 48 h later emission of fluorescence was recorded for CFP, FRET (left and middle panels,), and YFP channels to monitor photobleaching. FRET/CFP ratio signal (right panel) was recorded for cells in Ca 2+ -containing HBSS for 4–5 min. Calibration of FRET/CFP ratio to calculate Rmin and Rmax was performed individually for every assay. [Ca 2+ ] m data are presented as the mean ± s.d. of seven independent experiments

Techniques Used: Activity Assay, Transfection, Expressing, Fluorescence

Knockout of STIM1 expression by CRISPR/Cas9 D10A gene editing. a Strategy for gene editing using CRISPR/Cas9 D10A in SH-SY5Y cells. A pair of guide RNAs (sense and antisense) was designed to trigger a double nick at exon 5 of the STIM1 locus. PAM sequences are denoted in green font. Sequencing of a PCR product from the genomic DNA of the selected clone revealed a 211 + 318 base-pair insertion at the target site. The translated protein sequence is denoted in red font, with premature stop codons at the end of the sequences of both alleles. b The selected clone of cells was assessed for STIM1 expression by immunoblot, using two different anti-STIM1 antibodies generated against C-terminal and N-terminal epitopes. Anti-GAPDH antibody was used as loading control. c Ca 2+ entry was assessed as in Fig. 2 , i.e., triggering the emptying of intracellular stores with 1 μM Tg in Ca 2+ -free HBSS and adding 2 mM Ca 2+ back to the medium after store emptying. When required, the SOCE inhibitor BTP2 (3 μM) was added together with Tg. Data are presented as the mean ± s.d. of three independent experiments ( n = 85 cells for KO; n = 75 cells for wild-type; n = 42 cells for WT + BTP2). d Steady-state cytosolic free Ca 2+ concentration in WT and STIM1-KO cells. Left panel: Fura-2-loaded cells were incubated in Ca 2+ -containing HBSS (1.26 mM), and [Ca 2+ ] i was measured as indicated in the Methods section. After recording the F340/F380 ratio signal, the medium was replaced by Ca 2+ -free HBSS to evaluate the contribution of extracellular Ca 2+ entry to the [Ca 2+ ] i in resting conditions. Right panel: After calibration of the fura-2 signal, the [Ca 2+ ] i in resting conditions in Ca 2+ -containing HBSS was 77.4 ± 10 nM for wild-type cells, and 44 ± 4.4 nM in STIM1-KO cells. Data are the mean ± s.d. of three independent experiments ( n = number of cells for each condition)
Figure Legend Snippet: Knockout of STIM1 expression by CRISPR/Cas9 D10A gene editing. a Strategy for gene editing using CRISPR/Cas9 D10A in SH-SY5Y cells. A pair of guide RNAs (sense and antisense) was designed to trigger a double nick at exon 5 of the STIM1 locus. PAM sequences are denoted in green font. Sequencing of a PCR product from the genomic DNA of the selected clone revealed a 211 + 318 base-pair insertion at the target site. The translated protein sequence is denoted in red font, with premature stop codons at the end of the sequences of both alleles. b The selected clone of cells was assessed for STIM1 expression by immunoblot, using two different anti-STIM1 antibodies generated against C-terminal and N-terminal epitopes. Anti-GAPDH antibody was used as loading control. c Ca 2+ entry was assessed as in Fig. 2 , i.e., triggering the emptying of intracellular stores with 1 μM Tg in Ca 2+ -free HBSS and adding 2 mM Ca 2+ back to the medium after store emptying. When required, the SOCE inhibitor BTP2 (3 μM) was added together with Tg. Data are presented as the mean ± s.d. of three independent experiments ( n = 85 cells for KO; n = 75 cells for wild-type; n = 42 cells for WT + BTP2). d Steady-state cytosolic free Ca 2+ concentration in WT and STIM1-KO cells. Left panel: Fura-2-loaded cells were incubated in Ca 2+ -containing HBSS (1.26 mM), and [Ca 2+ ] i was measured as indicated in the Methods section. After recording the F340/F380 ratio signal, the medium was replaced by Ca 2+ -free HBSS to evaluate the contribution of extracellular Ca 2+ entry to the [Ca 2+ ] i in resting conditions. Right panel: After calibration of the fura-2 signal, the [Ca 2+ ] i in resting conditions in Ca 2+ -containing HBSS was 77.4 ± 10 nM for wild-type cells, and 44 ± 4.4 nM in STIM1-KO cells. Data are the mean ± s.d. of three independent experiments ( n = number of cells for each condition)

Techniques Used: Knock-Out, Expressing, CRISPR, Sequencing, Polymerase Chain Reaction, Generated, Concentration Assay, Incubation

3) Product Images from "Immunotherapeutic Targeting of Membrane Hsp70-Expressing Tumors Using Recombinant Human Granzyme B"

Article Title: Immunotherapeutic Targeting of Membrane Hsp70-Expressing Tumors Using Recombinant Human Granzyme B

Journal: PLoS ONE

doi: 10.1371/journal.pone.0041341

Comparative flow cytometric histograms of membrane Hsp70 expression on viable (7-AAD negative) cells from primary tumors and distant metastases of three patients, and on a relapse tumor and a distant metastases of another patient using FITC-labelled IgG1 isotype-matched control antibody (open histogram) and cmHsp70.1 mAb (grey histogram). The mean fluorescence intensity of Hsp70 is much higher on metastases compared to primary and relapse tumors, as indicated by a shift of the grey peak to the right.
Figure Legend Snippet: Comparative flow cytometric histograms of membrane Hsp70 expression on viable (7-AAD negative) cells from primary tumors and distant metastases of three patients, and on a relapse tumor and a distant metastases of another patient using FITC-labelled IgG1 isotype-matched control antibody (open histogram) and cmHsp70.1 mAb (grey histogram). The mean fluorescence intensity of Hsp70 is much higher on metastases compared to primary and relapse tumors, as indicated by a shift of the grey peak to the right.

Techniques Used: Flow Cytometry, Expressing, Fluorescence

4) Product Images from "Toll like receptor-3 ligand poly-ICLC promotes the efficacy of peripheral vaccinations with tumor antigen-derived peptide epitopes in murine CNS tumor models"

Article Title: Toll like receptor-3 ligand poly-ICLC promotes the efficacy of peripheral vaccinations with tumor antigen-derived peptide epitopes in murine CNS tumor models

Journal: Journal of Translational Medicine

doi: 10.1186/1479-5876-5-10

Poly-ICLC enhances the T-cell expression of α4-integrin (CD49d), which confers efficient CNS-tumor homing of Ag-specific T-cells . (A and B), C57BL/6 mice bearing day 10 i.c. M05 tumors received 5 × 10 6 naive OT-1 mouse-derived T-cells, then OVA-vaccines and/or poly-ICLC administrations on days 10 and 15. BILs (A) and SPCs (B) were harvested on day 16, and evaluated for the α 4 -integrin expression on CD8 + , OVA-tetramer + cells by flow cytometry. (A), numbers represent the percentage of α 4 -integrin + /CD8 + (upper panel), α 4 -integrin + /OVA + cells (lower panel) in lymphocyte-gated BIL populations. (B), numbers represent the percentage of α 4 -integrin + /OVA + cells in lymphocyte-gated SPC populations. (C), C57BL/6 mice bearing day 15 i.c. GL261 tumors received 5 × 10 6 naive Pmel-1 mouse-derived T cells, then hgp100-vaccines and/or poly-ICLC administrations on days 15 and 20. BILs were harvested on day 21, and evaluated for the α 4 -integrin expression on CD8 + /TCRvβ13 + cells by flow cytometry. Numbers represent the percentage of α 4 -integrin + /TCRvβ13 + T-cells in lymphocyte-gated populations. (D and E), mAb-mediated blockade of α4-integrin inhibited the CNS-tumor infiltration of OVA-specific T-cells, while not depleting Ag-reactive T-cells systemically. C57BL/6 mice bearing day 10 i.c. M05 tumors received i.p. injections of anti-α 4-integrin mAbs (R1-2, 150 μg/mouse and 9C10, 150 μg/mouse), or control isotype mAb (rat IgG 2b K, clone, A95-1, 300 μg/mouse) at 2 hrs before i.v. adoptive transfer of 5 × 10 6 naïve OT-1 mouse-derived T-cells and subsequent OVA-vaccination and poly-ICLC administration. On day 13, the mice received the 2 nd OVA-vaccination and poly-ICLC administration at 2 hrs following the 2 nd i.p. mAb injections. BILs, SPC and lymphocytes from draining inguinal (i)LNs were harvested on day 16. Numbers of CD3 + /OVA tetramer + BILs per mouse (D), and the presence of CD8 + , OVA-tetramer reactive T cells in iLN and SPC are depicted (E). Data are representative of 3 independent experiments with similar results.
Figure Legend Snippet: Poly-ICLC enhances the T-cell expression of α4-integrin (CD49d), which confers efficient CNS-tumor homing of Ag-specific T-cells . (A and B), C57BL/6 mice bearing day 10 i.c. M05 tumors received 5 × 10 6 naive OT-1 mouse-derived T-cells, then OVA-vaccines and/or poly-ICLC administrations on days 10 and 15. BILs (A) and SPCs (B) were harvested on day 16, and evaluated for the α 4 -integrin expression on CD8 + , OVA-tetramer + cells by flow cytometry. (A), numbers represent the percentage of α 4 -integrin + /CD8 + (upper panel), α 4 -integrin + /OVA + cells (lower panel) in lymphocyte-gated BIL populations. (B), numbers represent the percentage of α 4 -integrin + /OVA + cells in lymphocyte-gated SPC populations. (C), C57BL/6 mice bearing day 15 i.c. GL261 tumors received 5 × 10 6 naive Pmel-1 mouse-derived T cells, then hgp100-vaccines and/or poly-ICLC administrations on days 15 and 20. BILs were harvested on day 21, and evaluated for the α 4 -integrin expression on CD8 + /TCRvβ13 + cells by flow cytometry. Numbers represent the percentage of α 4 -integrin + /TCRvβ13 + T-cells in lymphocyte-gated populations. (D and E), mAb-mediated blockade of α4-integrin inhibited the CNS-tumor infiltration of OVA-specific T-cells, while not depleting Ag-reactive T-cells systemically. C57BL/6 mice bearing day 10 i.c. M05 tumors received i.p. injections of anti-α 4-integrin mAbs (R1-2, 150 μg/mouse and 9C10, 150 μg/mouse), or control isotype mAb (rat IgG 2b K, clone, A95-1, 300 μg/mouse) at 2 hrs before i.v. adoptive transfer of 5 × 10 6 naïve OT-1 mouse-derived T-cells and subsequent OVA-vaccination and poly-ICLC administration. On day 13, the mice received the 2 nd OVA-vaccination and poly-ICLC administration at 2 hrs following the 2 nd i.p. mAb injections. BILs, SPC and lymphocytes from draining inguinal (i)LNs were harvested on day 16. Numbers of CD3 + /OVA tetramer + BILs per mouse (D), and the presence of CD8 + , OVA-tetramer reactive T cells in iLN and SPC are depicted (E). Data are representative of 3 independent experiments with similar results.

Techniques Used: Expressing, Mouse Assay, Derivative Assay, Flow Cytometry, Cytometry, Adoptive Transfer Assay

5) Product Images from "Physiological pathway of differentiation of hematopoietic stem cell population into mural cells"

Article Title: Physiological pathway of differentiation of hematopoietic stem cell population into mural cells

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20050373

Induction of differentiation of the HSC/HPC population existing in the fetal liver into MCs. (a) FACS analysis of the HSC/HPC population existing in the mouse fetal liver at E12.5. The number in the top-right quadrant indicates the percentage of c-Kit + CD45 + cells among Lin − cells. The data is representative of three independent experiments. (b–f) The HSC/HPC population indicated by the box in panel a was cultured with or without cells from the brain in the presence of PDGF-BB and VEGF. (b) HSCs/HPCs alone cultured for 14 d were stained with anti-SMA mAb. (c) Schematic presentation of the coculture system with HSCs/HPCs from the fetal liver and cells from the brains of E12.5 mouse embryos. (d and e) HSCs/HPCs cultured with cells from the brain were stained with anti-SMA mAb (brown; d) or anti–CD31 mAb (dark blue; e). Bar in panel e, 30 μm. (f) Growth factors affecting the induction of HSCs/HPCs into MCs or ECs. HSCs/HPCs indicated by the box in panel a were cultured in the basic medium alone (EBM2), in EBM2 with 0.5 or 5 ng/ml TGF-β or 10 ng/ml bFGF, or with cells from the brain as described above. Cells adhering to culture plates were stained with anti–CD31 mAb or anti-SMA mAb, and the number of positively stained cells were determined. Results are expressed as the mean ± SD ( n = 5).
Figure Legend Snippet: Induction of differentiation of the HSC/HPC population existing in the fetal liver into MCs. (a) FACS analysis of the HSC/HPC population existing in the mouse fetal liver at E12.5. The number in the top-right quadrant indicates the percentage of c-Kit + CD45 + cells among Lin − cells. The data is representative of three independent experiments. (b–f) The HSC/HPC population indicated by the box in panel a was cultured with or without cells from the brain in the presence of PDGF-BB and VEGF. (b) HSCs/HPCs alone cultured for 14 d were stained with anti-SMA mAb. (c) Schematic presentation of the coculture system with HSCs/HPCs from the fetal liver and cells from the brains of E12.5 mouse embryos. (d and e) HSCs/HPCs cultured with cells from the brain were stained with anti-SMA mAb (brown; d) or anti–CD31 mAb (dark blue; e). Bar in panel e, 30 μm. (f) Growth factors affecting the induction of HSCs/HPCs into MCs or ECs. HSCs/HPCs indicated by the box in panel a were cultured in the basic medium alone (EBM2), in EBM2 with 0.5 or 5 ng/ml TGF-β or 10 ng/ml bFGF, or with cells from the brain as described above. Cells adhering to culture plates were stained with anti–CD31 mAb or anti-SMA mAb, and the number of positively stained cells were determined. Results are expressed as the mean ± SD ( n = 5).

Techniques Used: FACS, Cell Culture, Staining

CD45 + CD11b + cells existing in the PB under the hypoxia differentiate into MCs. (A) FACS analysis of HCs in the PB of mice. Mononuclear cells dual stained with anti-CD45 mAb and anti-CD11b mAb under normoxic (steady-state) conditions and ischemia. The numbers indicate the fraction of the cells in each box in the total mononuclear cells in the PB. Cells fractionated as CD45 + CD11b low (b) and CD45 + CD11b high (c) were sorted separately and cultured in the presence of VEGF and PDGF-BB. Cultured cells on fibronectin-coated plates were stained with anti-SMA mAb (brown) and anti–Flk-1 mAb (dark blue). Bar, 25 μm. (B) Development of CD31 + ECs and SMA + SMCs from CD11b high and CD11b low cells in the PB of mice in which ischemia had been induced by ligation of the femoral artery. CD11 + cells were collected from the PB on days 1,3, and 7 after ischemia was induced and cultured for 14 d. (C) Time course of EC (left) and MC (right) development from CD11b low cells (green square) or CD11b high cells (red circle). Results are expressed as the mean ± SD ( n = 5). (D) Limiting dilution analysis of CD11b low cells (green square) or CD11b high cells (red circle) for differentiation into ECs (a) and MCs (b). The frequency of cell differentiation into ECs or MCs is depicted on the graph based on the Poisson analysis.
Figure Legend Snippet: CD45 + CD11b + cells existing in the PB under the hypoxia differentiate into MCs. (A) FACS analysis of HCs in the PB of mice. Mononuclear cells dual stained with anti-CD45 mAb and anti-CD11b mAb under normoxic (steady-state) conditions and ischemia. The numbers indicate the fraction of the cells in each box in the total mononuclear cells in the PB. Cells fractionated as CD45 + CD11b low (b) and CD45 + CD11b high (c) were sorted separately and cultured in the presence of VEGF and PDGF-BB. Cultured cells on fibronectin-coated plates were stained with anti-SMA mAb (brown) and anti–Flk-1 mAb (dark blue). Bar, 25 μm. (B) Development of CD31 + ECs and SMA + SMCs from CD11b high and CD11b low cells in the PB of mice in which ischemia had been induced by ligation of the femoral artery. CD11 + cells were collected from the PB on days 1,3, and 7 after ischemia was induced and cultured for 14 d. (C) Time course of EC (left) and MC (right) development from CD11b low cells (green square) or CD11b high cells (red circle). Results are expressed as the mean ± SD ( n = 5). (D) Limiting dilution analysis of CD11b low cells (green square) or CD11b high cells (red circle) for differentiation into ECs (a) and MCs (b). The frequency of cell differentiation into ECs or MCs is depicted on the graph based on the Poisson analysis.

Techniques Used: FACS, Mouse Assay, Staining, Cell Culture, Ligation, Cell Differentiation

HSC population in the embryonic brain can give rise to MCs. (a–h) Brain sections at E10.5 (a–c), E11.5 (d–f), and E12.5 (g and h) from wild-type mice (a, b, d, e, g, and h), and AML1 mutant mice (c and f) were dual stained with anti–c-Kit mAb (red) and anti-CD31 mAb (dark blue; a, d, and g) or with anti-SMA mAb (brown) and anti-CD31 (dark blue; b, c, e, f, and h). Inset (e and h) shows high power view indicated by box. Bar in panel a, 50 μm. (i) FACS analysis of HSC/HPC population existing in the mouse brain at E12.5. The number in the top-right quadrant indicates the percentage of c-Kit + CD45 + cells among Lin − cells. The data is representative of three independent experiments. (j–l) Phenotype of cultured HSC/HPC populations in vitro. The HSC/HPC population indicated by the box in panel i was cultured on fibronectin-coated culture plates for 14 d and stained with anti-CD31 (dark blue; j), anti-SMA mAb (brown; k and l), anti–PECAM-1 mAb (dark blue; k), or anti–Flk-1 mAb (dark blue; l). Arrows (k and l) indicate cells expressing CD31 or Flk-1, respectively. Bar in panel j, 30 μm.
Figure Legend Snippet: HSC population in the embryonic brain can give rise to MCs. (a–h) Brain sections at E10.5 (a–c), E11.5 (d–f), and E12.5 (g and h) from wild-type mice (a, b, d, e, g, and h), and AML1 mutant mice (c and f) were dual stained with anti–c-Kit mAb (red) and anti-CD31 mAb (dark blue; a, d, and g) or with anti-SMA mAb (brown) and anti-CD31 (dark blue; b, c, e, f, and h). Inset (e and h) shows high power view indicated by box. Bar in panel a, 50 μm. (i) FACS analysis of HSC/HPC population existing in the mouse brain at E12.5. The number in the top-right quadrant indicates the percentage of c-Kit + CD45 + cells among Lin − cells. The data is representative of three independent experiments. (j–l) Phenotype of cultured HSC/HPC populations in vitro. The HSC/HPC population indicated by the box in panel i was cultured on fibronectin-coated culture plates for 14 d and stained with anti-CD31 (dark blue; j), anti-SMA mAb (brown; k and l), anti–PECAM-1 mAb (dark blue; k), or anti–Flk-1 mAb (dark blue; l). Arrows (k and l) indicate cells expressing CD31 or Flk-1, respectively. Bar in panel j, 30 μm.

Techniques Used: Mouse Assay, Mutagenesis, Staining, FACS, Cell Culture, In Vitro, Expressing

Development of CD45 + CD11b + cells into ECs and MCs in newly developed blood vessels of ischemic thigh muscles. Differentiation into ECs (A) or MCs (B) of CD45 + CD11b − or CD45 + CD11b + cells injected into ischemic thigh muscles as described in Fig. 4 was observed using laser-scanning confocal microscopy. (A) Section from a thigh muscle injected with CD45 + CD11b + (a–c) or CD45 + CD11b − (d–f) cells from green mice. (a and d) GFP (green), (b and e) anti-CD31 mAb (red), (c) merged images of panels a and b, (f) merged image of panels d and e. Arrowheads indicate GFP + CD31 + ECs. Insets show high power view of area indicated by box. Bar, 50 μm. (B) Section from a thigh muscle injected with CD45 + CD11b + (a–c) or CD45 + CD11b − (d–f) cells from green mice. (a and d) GFP (green), (b and e) anti-PDGFRβ mAb (red), (c) merged image of panels a and b, (f) merged image of panels d and e. Arrowheads indicate GFP + PDGFRβ + MCs. Insets show high power view of area indicated by box. Bar, 50 μm. (C) Development of CD11b high or CD11b low cells into vascular cell in the ischemic thigh muscle at 2 wk (wk.s) or 6 mo (mo.s) after injection of cells. Percentage of vascular cells originating from CD11b high or CD11b low cells was expressed as percentage of total capillaries counted in 30 random fields. In brief, when the GFP signal was detected in a cross section of a capillary as seen in the insets of A (a–c), this capillary was counted as being formed by the injected cells.
Figure Legend Snippet: Development of CD45 + CD11b + cells into ECs and MCs in newly developed blood vessels of ischemic thigh muscles. Differentiation into ECs (A) or MCs (B) of CD45 + CD11b − or CD45 + CD11b + cells injected into ischemic thigh muscles as described in Fig. 4 was observed using laser-scanning confocal microscopy. (A) Section from a thigh muscle injected with CD45 + CD11b + (a–c) or CD45 + CD11b − (d–f) cells from green mice. (a and d) GFP (green), (b and e) anti-CD31 mAb (red), (c) merged images of panels a and b, (f) merged image of panels d and e. Arrowheads indicate GFP + CD31 + ECs. Insets show high power view of area indicated by box. Bar, 50 μm. (B) Section from a thigh muscle injected with CD45 + CD11b + (a–c) or CD45 + CD11b − (d–f) cells from green mice. (a and d) GFP (green), (b and e) anti-PDGFRβ mAb (red), (c) merged image of panels a and b, (f) merged image of panels d and e. Arrowheads indicate GFP + PDGFRβ + MCs. Insets show high power view of area indicated by box. Bar, 50 μm. (C) Development of CD11b high or CD11b low cells into vascular cell in the ischemic thigh muscle at 2 wk (wk.s) or 6 mo (mo.s) after injection of cells. Percentage of vascular cells originating from CD11b high or CD11b low cells was expressed as percentage of total capillaries counted in 30 random fields. In brief, when the GFP signal was detected in a cross section of a capillary as seen in the insets of A (a–c), this capillary was counted as being formed by the injected cells.

Techniques Used: Injection, Confocal Microscopy, Mouse Assay

6) Product Images from "Functional impairment of PRRSV-specific peripheral CD3+CD8high cells"

Article Title: Functional impairment of PRRSV-specific peripheral CD3+CD8high cells

Journal: Veterinary Research

doi: 10.1051/vetres/2009029

Time course of proportions of CD3 + CD8 high cells in PBMC that were derived from virus-infected pigs and that were in vitro restimulated with virus or mock-control. A shows data for PRRSV-infected pigs; B for PRV Begonia-infected pigs. Full symbols represent individual pig values after restimulation with virus (■ pig 1, ● pig 2, ▲ pig 3, ♦ pig 4, pig 5); empty symbols represent individual pig values after mock-restimulation (□ pig 1, O pig 2, ∆ pig 3, ◊ pig 4, x pig 5). Full lines represent average values of the pigs obtained after restimulation with virus. Dashed lines represent average values for the pigs obtained after mock-restimulation.
Figure Legend Snippet: Time course of proportions of CD3 + CD8 high cells in PBMC that were derived from virus-infected pigs and that were in vitro restimulated with virus or mock-control. A shows data for PRRSV-infected pigs; B for PRV Begonia-infected pigs. Full symbols represent individual pig values after restimulation with virus (■ pig 1, ● pig 2, ▲ pig 3, ♦ pig 4, pig 5); empty symbols represent individual pig values after mock-restimulation (□ pig 1, O pig 2, ∆ pig 3, ◊ pig 4, x pig 5). Full lines represent average values of the pigs obtained after restimulation with virus. Dashed lines represent average values for the pigs obtained after mock-restimulation.

Techniques Used: Derivative Assay, Infection, In Vitro

7) Product Images from "Normal Thymocyte Negative Selection in TRAIL-deficient Mice"

Article Title: Normal Thymocyte Negative Selection in TRAIL-deficient Mice

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20030634

Thymocyte apoptosis in vitro. Thymocytes from BALB/c and BALB/c TRAIL −/− E18 mice were cultured in plates coated with anti-CD3ɛ (145–2C11) plus anti-CD28 (37.51) mAbs or PBS. Neutralizing anti-TRAIL antibodies were added to some of the cultures. After 20 h, live and apoptotic cells were discriminated by staining with annexin V-FITC plus propidium iodide and flow cytometric analysis. Data represent mean and standard error of the mean for six BALB/c mice plus or minus the addition of anti-mTRAIL mAb) or four BALB/c TRAIL −/− mice.
Figure Legend Snippet: Thymocyte apoptosis in vitro. Thymocytes from BALB/c and BALB/c TRAIL −/− E18 mice were cultured in plates coated with anti-CD3ɛ (145–2C11) plus anti-CD28 (37.51) mAbs or PBS. Neutralizing anti-TRAIL antibodies were added to some of the cultures. After 20 h, live and apoptotic cells were discriminated by staining with annexin V-FITC plus propidium iodide and flow cytometric analysis. Data represent mean and standard error of the mean for six BALB/c mice plus or minus the addition of anti-mTRAIL mAb) or four BALB/c TRAIL −/− mice.

Techniques Used: In Vitro, Mouse Assay, Cell Culture, Staining, Flow Cytometry

8) Product Images from "In Vitro Generation of Interleukin 10-producing Regulatory CD4+ T Cells Is Induced by Immunosuppressive Drugs and Inhibited by T Helper Type 1 (Th1)- and Th2-inducing Cytokines"

Article Title: In Vitro Generation of Interleukin 10-producing Regulatory CD4+ T Cells Is Induced by Immunosuppressive Drugs and Inhibited by T Helper Type 1 (Th1)- and Th2-inducing Cytokines

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20011629

VitD3/Dex induces the development of T cells producing IL-10 only and no inflammatory cytokines upon neutralization of Th1 and Th2 polarizing cytokines. (A) Purified OVA-specific naive CD4 + T cells were activated under the same conditions as in Fig. 1 ; except that neutralizing anti–IL-12, anti–IFN-γ, and anti–IL-4 mAbs were also added. After three rounds of stimulation, cells were characterized for cytokine production by intracellular flow cytometric analysis. Identical results were obtained when T cells were isolated from DO11.10 RAG −/− . Representative results of more than five experiments are shown. (B) Purified naive CD4 + T cells from DO11.10 RAG −/− mice were activated using anti-CD3 and anti-CD28 stimulation under neutral or VitD3/Dex conditions. After three rounds of stimulation, cells were characterized for cytokine production by intracellular flow cytometric analysis. Identical results were obtained using DO11.10 or BALB/c mice. Representative results of three experiments are shown.
Figure Legend Snippet: VitD3/Dex induces the development of T cells producing IL-10 only and no inflammatory cytokines upon neutralization of Th1 and Th2 polarizing cytokines. (A) Purified OVA-specific naive CD4 + T cells were activated under the same conditions as in Fig. 1 ; except that neutralizing anti–IL-12, anti–IFN-γ, and anti–IL-4 mAbs were also added. After three rounds of stimulation, cells were characterized for cytokine production by intracellular flow cytometric analysis. Identical results were obtained when T cells were isolated from DO11.10 RAG −/− . Representative results of more than five experiments are shown. (B) Purified naive CD4 + T cells from DO11.10 RAG −/− mice were activated using anti-CD3 and anti-CD28 stimulation under neutral or VitD3/Dex conditions. After three rounds of stimulation, cells were characterized for cytokine production by intracellular flow cytometric analysis. Identical results were obtained using DO11.10 or BALB/c mice. Representative results of three experiments are shown.

Techniques Used: Neutralization, Purification, Flow Cytometry, Isolation, Mouse Assay

IL-10 is a positive autocrine factor to enhance the development of IL-10–producing T cells induced by Vit/Dex. (A) Cells were activated as in Fig. 2 in the combination of Vit/Dex, plus neutralizing anti–IL-12, anti–IFN-γ, and anti–IL-4 mAbs in the absence or presence of either anti–IL-10R (10 μg/ml) or anti–TGF-β (10 μg/ml). Additionally, T cells were stimulated using anti-CD3 and anti-CD28 with Vit/Dex in the absence (B) or presence of neutralizing anti–IFN-γ and anti–IL-4 mAbs (C), in the presence or absence of anti–IL-10R mAbs. After three rounds of stimulation, cells were characterized for cytokine production by intracellular flow cytometric analysis. Representative results of five experiments are shown.
Figure Legend Snippet: IL-10 is a positive autocrine factor to enhance the development of IL-10–producing T cells induced by Vit/Dex. (A) Cells were activated as in Fig. 2 in the combination of Vit/Dex, plus neutralizing anti–IL-12, anti–IFN-γ, and anti–IL-4 mAbs in the absence or presence of either anti–IL-10R (10 μg/ml) or anti–TGF-β (10 μg/ml). Additionally, T cells were stimulated using anti-CD3 and anti-CD28 with Vit/Dex in the absence (B) or presence of neutralizing anti–IFN-γ and anti–IL-4 mAbs (C), in the presence or absence of anti–IL-10R mAbs. After three rounds of stimulation, cells were characterized for cytokine production by intracellular flow cytometric analysis. Representative results of five experiments are shown.

Techniques Used: Flow Cytometry

VitD3/Dex induces the development of human T cells producing IL-10 and no IL-4, IL-5, or IFN-γ. Purified human CD4 + CD45RA + were stimulated with plate-bound anti-CD3, soluble anti-CD28, and IL-2 in the presence of neutralizing anti–IL-4, anti–IFN-γ, and anti–IL-12 mAbs. After four rounds of stimulation, cells were characterized for cytokine production by immunoassay (A) as well as by intracellular flow cytometric analysis (B). Representative results of four experiments are shown.
Figure Legend Snippet: VitD3/Dex induces the development of human T cells producing IL-10 and no IL-4, IL-5, or IFN-γ. Purified human CD4 + CD45RA + were stimulated with plate-bound anti-CD3, soluble anti-CD28, and IL-2 in the presence of neutralizing anti–IL-4, anti–IFN-γ, and anti–IL-12 mAbs. After four rounds of stimulation, cells were characterized for cytokine production by immunoassay (A) as well as by intracellular flow cytometric analysis (B). Representative results of four experiments are shown.

Techniques Used: Purification, Flow Cytometry

9) Product Images from "An anti-CD45RO/RB monoclonal antibody modulates T cell responses via induction of apoptosis and generation of regulatory T cells"

Article Title: An anti-CD45RO/RB monoclonal antibody modulates T cell responses via induction of apoptosis and generation of regulatory T cells

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20040912

ChA6 mAb treatment prolongs human islet allograft survival in hu-PBL-NOD/SCID recipient mice. (A) Diabetic NOD/SCID mice were transplanted under the kidney capsule with human islets. Mice were injected intraperitoneally with 50 × 10 6 allogeneic human PBMCs. Normal NOD/SCID mice (*, n = 8) were used as control of human islets function. Mice were treated with vehicle (•, n = 12); with the Edmonton protocol (♦, n = 4); chA6 mAb at days 0, 3, and 5 after transplantation (▪, n = 14); or sirolimus (▴, n = 3). Glycemia levels monitored graft survival. Asterisks indicate statistical analysis in which treated mice were compared with control mice (*** ≤ 0.0001). (B) Kidney bearing the human-islet graft from control, chA6 mAb–treated, or normal NOD/SCID mice at 100 d after transplantation were snap-frozen, and 5-μm-thick sections were stained with hematoxylin and eosin (HE). Alternatively, sections were stained for the expression of CD3 and insulin.
Figure Legend Snippet: ChA6 mAb treatment prolongs human islet allograft survival in hu-PBL-NOD/SCID recipient mice. (A) Diabetic NOD/SCID mice were transplanted under the kidney capsule with human islets. Mice were injected intraperitoneally with 50 × 10 6 allogeneic human PBMCs. Normal NOD/SCID mice (*, n = 8) were used as control of human islets function. Mice were treated with vehicle (•, n = 12); with the Edmonton protocol (♦, n = 4); chA6 mAb at days 0, 3, and 5 after transplantation (▪, n = 14); or sirolimus (▴, n = 3). Glycemia levels monitored graft survival. Asterisks indicate statistical analysis in which treated mice were compared with control mice (*** ≤ 0.0001). (B) Kidney bearing the human-islet graft from control, chA6 mAb–treated, or normal NOD/SCID mice at 100 d after transplantation were snap-frozen, and 5-μm-thick sections were stained with hematoxylin and eosin (HE). Alternatively, sections were stained for the expression of CD3 and insulin.

Techniques Used: Mouse Assay, Injection, Transplantation Assay, Staining, Expressing

ChA6 mAb inhibits allogeneic and TT-specific proliferation of CD4 + T cells. (A) Primary MLR: CD4 + T cells were stimulated with allogeneic CD3-depleted cells with or without chA6 mAb (10 μg/ml). Results from one representative donor out of 14 are shown. Secondary MLR: CD4 + T cells were primed with allogeneic CD3-depleted cells for 10 d. Cells were collected and washed, and proliferative responses to the same allogenic cells used in the primary stimulation in the presence or absence of chA6 mAb (10 μg/ml) were tested. Results from one representative donor out of four are shown. Dose-dependent inhibition of primary MLR: CD4 + T cells were stimulated with allogeneic CD3-depleted cells in the presence of the indicated concentrations of chA6 or chimeric isotype control mAbs. Results from one representative donor out of five are shown. R, responder; S, stimulator. (B) TT response: total PBMCs were stimulated with TT with or without chA6 mAb (10 μg/ml). Results from one representative donor out of 16 are shown. The percentages of inhibition of proliferation in the presence of chA6 mAb relative to control are indicated.
Figure Legend Snippet: ChA6 mAb inhibits allogeneic and TT-specific proliferation of CD4 + T cells. (A) Primary MLR: CD4 + T cells were stimulated with allogeneic CD3-depleted cells with or without chA6 mAb (10 μg/ml). Results from one representative donor out of 14 are shown. Secondary MLR: CD4 + T cells were primed with allogeneic CD3-depleted cells for 10 d. Cells were collected and washed, and proliferative responses to the same allogenic cells used in the primary stimulation in the presence or absence of chA6 mAb (10 μg/ml) were tested. Results from one representative donor out of four are shown. Dose-dependent inhibition of primary MLR: CD4 + T cells were stimulated with allogeneic CD3-depleted cells in the presence of the indicated concentrations of chA6 or chimeric isotype control mAbs. Results from one representative donor out of five are shown. R, responder; S, stimulator. (B) TT response: total PBMCs were stimulated with TT with or without chA6 mAb (10 μg/ml). Results from one representative donor out of 16 are shown. The percentages of inhibition of proliferation in the presence of chA6 mAb relative to control are indicated.

Techniques Used: Inhibition

ChA6 mAb induced MP.58-66–specific CD8 + T reg cells. (A) Enriched CD8 + T cells were stimulated with 10 μg/ml peptide MP.58-66, in the absence (MLP) or presence of 10 μg/ml chA6 mAb (MLPchA6). After two rounds of stimulation, MLP and MLPchA6 cells were rechallenged with T2 cells previously pulsed with the MP.58-66 peptide. After 24 h culture, IFN-γ was quantified in culture supernatants by ELISA. Results from one representative donor out of 16 are shown. The percentage of inhibition of IFN-γ release in the presence of chA6 mAb relative to control is presented. (B) MLP and MLPchA6 cells were stained with TCR-Vβ17–specific mAb. Results from one representative out of eight donors tested are shown. (C) After two rounds of stimulation, MLP cells were cocultured with increased amounts of MLPchA6 cells with MP.56-88 pulsed and nonpulsed T2 cells in the absence of chA6 mAb. After 24 h culture, IFN-γ was quantified in culture supernatants by ELISA. Results from one representative out of three donors tested are shown. The percentage of inhibition of IFN-γ release in the presence of chA6 mAb relative to control is presented. (D) MP.58-66–specific T cell lines were stained with the indicated surface molecules and analyzed by flow cytometry. Percentages of positive cells, set according to the isotype controls, are shown in the relative quadrant. Results from one representative donor out of four donors tested are shown. (E) MLP cells were cocultured with MLPchA6 cells (1:1 ratio) and stimulated with MP.56-88 pulsed and nonpulsed APC in the presence of 10 μg/ml anti-IL-10R and 10 μg/ml anti-TGF-β mAb. After 24 h of culture, IFN-γ was quantified in culture supernatants by ELISA. Results from one representative out of two donors tested are shown. The percentages of inhibition of IFN-γ release in the presence of chA6 mAb relative to control are presented.
Figure Legend Snippet: ChA6 mAb induced MP.58-66–specific CD8 + T reg cells. (A) Enriched CD8 + T cells were stimulated with 10 μg/ml peptide MP.58-66, in the absence (MLP) or presence of 10 μg/ml chA6 mAb (MLPchA6). After two rounds of stimulation, MLP and MLPchA6 cells were rechallenged with T2 cells previously pulsed with the MP.58-66 peptide. After 24 h culture, IFN-γ was quantified in culture supernatants by ELISA. Results from one representative donor out of 16 are shown. The percentage of inhibition of IFN-γ release in the presence of chA6 mAb relative to control is presented. (B) MLP and MLPchA6 cells were stained with TCR-Vβ17–specific mAb. Results from one representative out of eight donors tested are shown. (C) After two rounds of stimulation, MLP cells were cocultured with increased amounts of MLPchA6 cells with MP.56-88 pulsed and nonpulsed T2 cells in the absence of chA6 mAb. After 24 h culture, IFN-γ was quantified in culture supernatants by ELISA. Results from one representative out of three donors tested are shown. The percentage of inhibition of IFN-γ release in the presence of chA6 mAb relative to control is presented. (D) MP.58-66–specific T cell lines were stained with the indicated surface molecules and analyzed by flow cytometry. Percentages of positive cells, set according to the isotype controls, are shown in the relative quadrant. Results from one representative donor out of four donors tested are shown. (E) MLP cells were cocultured with MLPchA6 cells (1:1 ratio) and stimulated with MP.56-88 pulsed and nonpulsed APC in the presence of 10 μg/ml anti-IL-10R and 10 μg/ml anti-TGF-β mAb. After 24 h of culture, IFN-γ was quantified in culture supernatants by ELISA. Results from one representative out of two donors tested are shown. The percentages of inhibition of IFN-γ release in the presence of chA6 mAb relative to control are presented.

Techniques Used: Enzyme-linked Immunosorbent Assay, Inhibition, Staining, Flow Cytometry, Cytometry

ChA6 mAb induced TT-specific CD4 + T reg cells. (A) Total PBMCs were stimulated with TT with or without chA6 mAb (10 μg/ml). TT-specific cell lines were rechallenged with TT-pulsed and nonpulsed autologous irradiated monocytes in the absence of chA6 mAb. Results from one representative donor out of 18 are shown. The percentage of inhibition of proliferation in the presence of chA6 mAb relative to control is presented. Alternative: after 48 h culture, IFN-γ was quantified in culture supernatants by ELISA. Results from one representative donor out of 10 are shown. The percentage of inhibition of IFN-γ release in the presence of chA6 mAb relative to control is shown. Iono, ionomycin; TPA, 12- 0 -tetradecanoylphorbol-13-acetate. (B). TT-specific cell lines were cocultured with increased amounts of TT-specific T cell lines generated in the presence of chA6 mAb (TT/chA6) and stimulated with TT-pulsed and nonpulsed autologous irradiated monocytes in the absence of chA6 mAb. After 48 h of culture, IFN-γ was quantified in culture supernatants by ELISA. Results from one representative donor out of four are shown. The percentages of inhibition of proliferation in the presence of chA6 mAb relative to control are presented.
Figure Legend Snippet: ChA6 mAb induced TT-specific CD4 + T reg cells. (A) Total PBMCs were stimulated with TT with or without chA6 mAb (10 μg/ml). TT-specific cell lines were rechallenged with TT-pulsed and nonpulsed autologous irradiated monocytes in the absence of chA6 mAb. Results from one representative donor out of 18 are shown. The percentage of inhibition of proliferation in the presence of chA6 mAb relative to control is presented. Alternative: after 48 h culture, IFN-γ was quantified in culture supernatants by ELISA. Results from one representative donor out of 10 are shown. The percentage of inhibition of IFN-γ release in the presence of chA6 mAb relative to control is shown. Iono, ionomycin; TPA, 12- 0 -tetradecanoylphorbol-13-acetate. (B). TT-specific cell lines were cocultured with increased amounts of TT-specific T cell lines generated in the presence of chA6 mAb (TT/chA6) and stimulated with TT-pulsed and nonpulsed autologous irradiated monocytes in the absence of chA6 mAb. After 48 h of culture, IFN-γ was quantified in culture supernatants by ELISA. Results from one representative donor out of four are shown. The percentages of inhibition of proliferation in the presence of chA6 mAb relative to control are presented.

Techniques Used: Irradiation, Inhibition, Enzyme-linked Immunosorbent Assay, Generated

Dose-dependent apoptosis induced in CD4 + A6 bright T cells by chA6 mAb. (A) CD4 + T cells were incubated with the indicated concentrations of chA6 or isotype control mAbs. Cells were cultured overnight without (unstimulated) or with (stimulated) coated anti-CD3 (0.1 μg/ml) and soluble anti-CD28 (1 μg/ml) mAb and were analyzed for apoptosis. The percentages of early apoptotic (annexin V + /PI − ) cells and mean ± SE are shown. P values were calculated by t test: * P , effect of chA6 mAb on total CD4 + T cells; $ P , effect on annexin V–depleted CD4 + T cells (* P or $ P ≤ 0.05; ** P ≤ 0.005). (B) PBMCs of three healthy donors were cultured overnight in the presence of the indicated concentrations of chA6 mAb (filled symbols) or an IgG1 isotype control antibody (open symbols) and analyzed for apoptosis. Curve fitting and ED 50 value calculations were performed. (C) Total or annexin V–depleted CD4 + T cells were incubated with the indicated concentrations of chA6 mAb. Cells were cultured overnight without or with (stimulated) coated anti-CD3 (0.1 μg/ml) and soluble anti-CD28 (1 μg/ml) mAbs and were stained with annexin V FITC mAb and chA6 mAb followed by anti-human IgG1PE mAb and analyzed by flow cytometry. Percentages of positive cells, set according to the isotype-matched controls (not shown), are shown in the top corner of the quadrant. Results from one representative out of 10 different donors tested are shown. (D) CD4 + T cells were incubated with the indicated concentrations of anti-CD45RO (UCLH-1), anti-CD45RA (HI-100), chA6, or isotype control mAbs. Cells were stimulated with coated anti-CD3 (0.1 μg/ml) and soluble anti-CD28 (1 μg/ml) mAbs, cultured overnight, and analyzed for apoptosis. The percentages of early-apoptotic (annexinV + /PI − ) cells are shown. (E) Total or annexin V–depleted CD4 + T cells were stimulated with allogeneic CD3-depleted cells with or without chA6 mAb (10 μg/ml). Results from one representative donor out of four are shown. The percentages of inhibition of proliferation in the presence of chA6 mAb relative to control are presented.
Figure Legend Snippet: Dose-dependent apoptosis induced in CD4 + A6 bright T cells by chA6 mAb. (A) CD4 + T cells were incubated with the indicated concentrations of chA6 or isotype control mAbs. Cells were cultured overnight without (unstimulated) or with (stimulated) coated anti-CD3 (0.1 μg/ml) and soluble anti-CD28 (1 μg/ml) mAb and were analyzed for apoptosis. The percentages of early apoptotic (annexin V + /PI − ) cells and mean ± SE are shown. P values were calculated by t test: * P , effect of chA6 mAb on total CD4 + T cells; $ P , effect on annexin V–depleted CD4 + T cells (* P or $ P ≤ 0.05; ** P ≤ 0.005). (B) PBMCs of three healthy donors were cultured overnight in the presence of the indicated concentrations of chA6 mAb (filled symbols) or an IgG1 isotype control antibody (open symbols) and analyzed for apoptosis. Curve fitting and ED 50 value calculations were performed. (C) Total or annexin V–depleted CD4 + T cells were incubated with the indicated concentrations of chA6 mAb. Cells were cultured overnight without or with (stimulated) coated anti-CD3 (0.1 μg/ml) and soluble anti-CD28 (1 μg/ml) mAbs and were stained with annexin V FITC mAb and chA6 mAb followed by anti-human IgG1PE mAb and analyzed by flow cytometry. Percentages of positive cells, set according to the isotype-matched controls (not shown), are shown in the top corner of the quadrant. Results from one representative out of 10 different donors tested are shown. (D) CD4 + T cells were incubated with the indicated concentrations of anti-CD45RO (UCLH-1), anti-CD45RA (HI-100), chA6, or isotype control mAbs. Cells were stimulated with coated anti-CD3 (0.1 μg/ml) and soluble anti-CD28 (1 μg/ml) mAbs, cultured overnight, and analyzed for apoptosis. The percentages of early-apoptotic (annexinV + /PI − ) cells are shown. (E) Total or annexin V–depleted CD4 + T cells were stimulated with allogeneic CD3-depleted cells with or without chA6 mAb (10 μg/ml). Results from one representative donor out of four are shown. The percentages of inhibition of proliferation in the presence of chA6 mAb relative to control are presented.

Techniques Used: Incubation, Cell Culture, Staining, Flow Cytometry, Cytometry, Inhibition

ChA6 mAb induce apoptosis in CD4 + T cells through the activation of the intrinsic pathway. (A) CD4 + T cells were incubated with the indicated concentrations of chA6 mAb. Cells were cultured overnight with or without coated anti-CD3 (0.1 μg/ml) and soluble anti-CD28 (1 μg/ml) mAbs. Western blot tests with anti-caspase-3, anti-caspase-8, and anti-caspase-9 mAb were performed. As positive control, Hela cells were treated with staurosporine, 5 μM. Amounts of loaded proteins have been controlled for homogeneity by probing membranes with an anti- β-actin mAb. Weak expression of the cleavage products of caspase-9 in CD4 + T cells cultured in medium is considered background apoptosis. Results from one representative donor out of three are shown. (B) CD4 + T cells were incubated with chA6 mAb (5 μg/ml) in the presence of 15 μg/ml of cross-linking goat anti–human IgG (F(ab′) 2 ). Mitochondrial-based death pathways were assessed using 3,3′-dihexyloxacarbocyanine iodide (DiOC 6 (3)) accumulation that reflects changes in Δψm in the mitochondria. Results from one representative donor out of three are shown.
Figure Legend Snippet: ChA6 mAb induce apoptosis in CD4 + T cells through the activation of the intrinsic pathway. (A) CD4 + T cells were incubated with the indicated concentrations of chA6 mAb. Cells were cultured overnight with or without coated anti-CD3 (0.1 μg/ml) and soluble anti-CD28 (1 μg/ml) mAbs. Western blot tests with anti-caspase-3, anti-caspase-8, and anti-caspase-9 mAb were performed. As positive control, Hela cells were treated with staurosporine, 5 μM. Amounts of loaded proteins have been controlled for homogeneity by probing membranes with an anti- β-actin mAb. Weak expression of the cleavage products of caspase-9 in CD4 + T cells cultured in medium is considered background apoptosis. Results from one representative donor out of three are shown. (B) CD4 + T cells were incubated with chA6 mAb (5 μg/ml) in the presence of 15 μg/ml of cross-linking goat anti–human IgG (F(ab′) 2 ). Mitochondrial-based death pathways were assessed using 3,3′-dihexyloxacarbocyanine iodide (DiOC 6 (3)) accumulation that reflects changes in Δψm in the mitochondria. Results from one representative donor out of three are shown.

Techniques Used: Activation Assay, Incubation, Cell Culture, Western Blot, Positive Control, Expressing

ChA6 mAb inhibits polyclonal proliferation of CD4 + T cells. (A) CD4 + T cells were stimulated with the indicated concentrations of coated anti-CD3 mAb in the presence or absence of chA6 or chimeric isotype control mAb (10 μg/ml) with or without soluble anti-CD28 mAb (1 μg/ml). Results from one representative donor out of four are shown. (B) Dose-dependent inhibition of proliferative polyclonal T cell response by chA6 mAb. CD4 + T cells were stimulated with 0.1 and 0.01 μg/ml of coated anti-CD3 mAb in the presence of the indicated concentrations of soluble chA6 mAb. Results from one representative donor out of three (0.1 μg/ml of anti-CD3 mAb) and out of two (0.01 μg/ml of anti-CD3 mAb) are shown. The percentages of inhibition of proliferation in the presence of chA6 mAb relative to control are indicated.
Figure Legend Snippet: ChA6 mAb inhibits polyclonal proliferation of CD4 + T cells. (A) CD4 + T cells were stimulated with the indicated concentrations of coated anti-CD3 mAb in the presence or absence of chA6 or chimeric isotype control mAb (10 μg/ml) with or without soluble anti-CD28 mAb (1 μg/ml). Results from one representative donor out of four are shown. (B) Dose-dependent inhibition of proliferative polyclonal T cell response by chA6 mAb. CD4 + T cells were stimulated with 0.1 and 0.01 μg/ml of coated anti-CD3 mAb in the presence of the indicated concentrations of soluble chA6 mAb. Results from one representative donor out of three (0.1 μg/ml of anti-CD3 mAb) and out of two (0.01 μg/ml of anti-CD3 mAb) are shown. The percentages of inhibition of proliferation in the presence of chA6 mAb relative to control are indicated.

Techniques Used: Inhibition

ChA6 mAb induced TT-specific CD4 + T reg 1 cells. (A) TT-specific cell lines were stained with the indicated surface molecules and analyzed by flow cytometry with gating on live CD4 + T cells. Thin lines represent staining with the appropriate control mAbs. Results from one representative donor out of 10 are shown. (B) TT and TT/chA6 cell lines were stimulated with TT-pulsed autologous irradiated monocytes in the absence of chA6 mAb. Supernatants were collected after 72 h, and levels of IL-10 and TGF-β were determined by ELISA. (C) TT cell lines were cocultured with TT/chA6 cell lines (1:1 ratio) and stimulated with TT-pulsed and nonpulsed autologous irradiated monocytes in the presence of 10 μg/ml of anti–IL-10R mAb. After 48 h culture, IFN-γ was quantified in culture supernatants by ELISA. Results from one representative donor out of two are shown. The percentage of inhibition of IFN-γ release in the presence of chA6 mAb relative to control is presented.
Figure Legend Snippet: ChA6 mAb induced TT-specific CD4 + T reg 1 cells. (A) TT-specific cell lines were stained with the indicated surface molecules and analyzed by flow cytometry with gating on live CD4 + T cells. Thin lines represent staining with the appropriate control mAbs. Results from one representative donor out of 10 are shown. (B) TT and TT/chA6 cell lines were stimulated with TT-pulsed autologous irradiated monocytes in the absence of chA6 mAb. Supernatants were collected after 72 h, and levels of IL-10 and TGF-β were determined by ELISA. (C) TT cell lines were cocultured with TT/chA6 cell lines (1:1 ratio) and stimulated with TT-pulsed and nonpulsed autologous irradiated monocytes in the presence of 10 μg/ml of anti–IL-10R mAb. After 48 h culture, IFN-γ was quantified in culture supernatants by ELISA. Results from one representative donor out of two are shown. The percentage of inhibition of IFN-γ release in the presence of chA6 mAb relative to control is presented.

Techniques Used: Staining, Flow Cytometry, Cytometry, Irradiation, Enzyme-linked Immunosorbent Assay, Inhibition

10) Product Images from "Neutrophils Induced Licensing of Natural Killer Cells"

Article Title: Neutrophils Induced Licensing of Natural Killer Cells

Journal: Mediators of Inflammation

doi: 10.1155/2015/747680

Neutrophils promote a licensing effect of NK cells: upregulation of intracellular IFN- γ expression. Forty Gy-irradiated 2 × 10 6 mononuclear cells (PB and G-PB) of B10.D2 mice were injected intraperitoneally into B10 mice. Two days after injection, PBMCs of recipient B10 mice were analyzed by flow cytometry. Representative dot plots show intracellular IFN- γ expressions gated on DX5 + CD3 − Ly49G2 + NK cells ((a), upper panel) and DX5 + CD3 − Ly49C/I + NK cells ((a), lower panel) in uninjected B10 PBMCs (control), B10 PBMCs injected by B10.D2 PBMCs (PB), and B10 PBMCs injected by G-CSF-treated B10.D2 PBMCs (G-PB). The comparison of IFN- γ expression of DX5 + CD3 − Ly49G2 + NK cells and DX5 + CD3 − Ly49C/I + NK cells in control ( n = 4), PB ( n = 4), and G-PB ( n = 4) was depicted (b). Data are expressed as the means ± SD. ∗ P
Figure Legend Snippet: Neutrophils promote a licensing effect of NK cells: upregulation of intracellular IFN- γ expression. Forty Gy-irradiated 2 × 10 6 mononuclear cells (PB and G-PB) of B10.D2 mice were injected intraperitoneally into B10 mice. Two days after injection, PBMCs of recipient B10 mice were analyzed by flow cytometry. Representative dot plots show intracellular IFN- γ expressions gated on DX5 + CD3 − Ly49G2 + NK cells ((a), upper panel) and DX5 + CD3 − Ly49C/I + NK cells ((a), lower panel) in uninjected B10 PBMCs (control), B10 PBMCs injected by B10.D2 PBMCs (PB), and B10 PBMCs injected by G-CSF-treated B10.D2 PBMCs (G-PB). The comparison of IFN- γ expression of DX5 + CD3 − Ly49G2 + NK cells and DX5 + CD3 − Ly49C/I + NK cells in control ( n = 4), PB ( n = 4), and G-PB ( n = 4) was depicted (b). Data are expressed as the means ± SD. ∗ P

Techniques Used: Expressing, Irradiation, Mouse Assay, Injection, Flow Cytometry, Cytometry

Enrichment and depletion of neutrophils affect licensing effect of NK cells. B10.D2 PB was enriched or depleted of CD11b + Gr-1 + neutrophils by magnet beads negative selection (a). Forty Gy-irradiated 2 × 10 6 PBMCs or neutrophil-enriched/depleted cells were injected intraperitoneally into B10 mice. Two days after injection, PBMCs of recipient B10 mice were analyzed by flow cytometry. Representative dot plot analysis showed intracellular IFN- γ expressions gated on DX5 + CD3 − Ly49G2 + NK cells ((b), upper panel) and DX5 + CD3 − Ly49C/I + NK cells ((b), lower panel) in uninjected B10 PBMCs (control), B10 PBMCs injected by B10.D2 PBMCs (PB untreated), B10.D2 PBMCs enriched neutrophils (neutrophil rich), and B10.D2 PBMCs depleted of neutrophils (neutrophil depleted). The comparison of IFN- γ expression of DX5 + CD3 − Ly49G2 + NK cells and DX5 + CD3 − Ly49C/I + NK cells in control ( n = 6), PB untreated ( n = 9), neutrophil rich ( n = 4), and neutrophil depleted ( n = 4) was depicted (c). Data are expressed as the means ± SD. ∗ P
Figure Legend Snippet: Enrichment and depletion of neutrophils affect licensing effect of NK cells. B10.D2 PB was enriched or depleted of CD11b + Gr-1 + neutrophils by magnet beads negative selection (a). Forty Gy-irradiated 2 × 10 6 PBMCs or neutrophil-enriched/depleted cells were injected intraperitoneally into B10 mice. Two days after injection, PBMCs of recipient B10 mice were analyzed by flow cytometry. Representative dot plot analysis showed intracellular IFN- γ expressions gated on DX5 + CD3 − Ly49G2 + NK cells ((b), upper panel) and DX5 + CD3 − Ly49C/I + NK cells ((b), lower panel) in uninjected B10 PBMCs (control), B10 PBMCs injected by B10.D2 PBMCs (PB untreated), B10.D2 PBMCs enriched neutrophils (neutrophil rich), and B10.D2 PBMCs depleted of neutrophils (neutrophil depleted). The comparison of IFN- γ expression of DX5 + CD3 − Ly49G2 + NK cells and DX5 + CD3 − Ly49C/I + NK cells in control ( n = 6), PB untreated ( n = 9), neutrophil rich ( n = 4), and neutrophil depleted ( n = 4) was depicted (c). Data are expressed as the means ± SD. ∗ P

Techniques Used: Selection, Irradiation, Injection, Mouse Assay, Flow Cytometry, Cytometry, Expressing

Neutrophils promote a licensing effect of NK cells: CD107a degranulation assay. Forty Gy-irradiated 2 × 10 6 mononuclear cells (splenocyte, SPL; peripheral blood, PB; and G-CSF-treated PB, G-PB) of B10.D2 mice were injected intraperitoneally into B10 mice. Two days after injection of cells, peripheral blood mononuclear cells of recipient B10 mice were analyzed by flow cytometry. Representative histograms show CD107a expressions gated on DX5 + CD3 − Ly49G2 + cells ((a), left) and DX5 + CD3 − Ly49C/I + cells ((a), right) in treated B10 mice (solid line) and untreated as controls (shadow area). Time course of mean fluorescence intensity (MFI) of CD107a expressions of DX5 + CD3 − Ly49G2 + cells ((b), left) and DX5 + CD3 − Ly49C/I + cells ((b), right) was analyzed before ( n = 11) and after injection of SPL (open circle, n = 18), PB (open triangle, n = 18), and G-PB (filled square, n = 8). Data are expressed as the means ± SD. ∗ P
Figure Legend Snippet: Neutrophils promote a licensing effect of NK cells: CD107a degranulation assay. Forty Gy-irradiated 2 × 10 6 mononuclear cells (splenocyte, SPL; peripheral blood, PB; and G-CSF-treated PB, G-PB) of B10.D2 mice were injected intraperitoneally into B10 mice. Two days after injection of cells, peripheral blood mononuclear cells of recipient B10 mice were analyzed by flow cytometry. Representative histograms show CD107a expressions gated on DX5 + CD3 − Ly49G2 + cells ((a), left) and DX5 + CD3 − Ly49C/I + cells ((a), right) in treated B10 mice (solid line) and untreated as controls (shadow area). Time course of mean fluorescence intensity (MFI) of CD107a expressions of DX5 + CD3 − Ly49G2 + cells ((b), left) and DX5 + CD3 − Ly49C/I + cells ((b), right) was analyzed before ( n = 11) and after injection of SPL (open circle, n = 18), PB (open triangle, n = 18), and G-PB (filled square, n = 8). Data are expressed as the means ± SD. ∗ P

Techniques Used: Degranulation Assay, Irradiation, Mouse Assay, Injection, Flow Cytometry, Cytometry, Fluorescence

11) Product Images from "Treadmill exercise induces murine cardiac allograft survival and generates regulatory T cell"

Article Title: Treadmill exercise induces murine cardiac allograft survival and generates regulatory T cell

Journal: Transplant International

doi: 10.1111/tri.12491

Evidence of generation of regulatory cells in treadmill-exercised CBA allograft recipients. (a) Scheme on adoptive transfer study to confirm the generation of regulatory T cells. (b, c) Cardiac allograft survival after adoptive transfer of whole splenocytes (b) or CD4 + cells (c). (d–h) Results of double immunostaining of cardiac allografts obtained 4 weeks after transplantation from untreated mice and postoperative 1-week treadmill-exercised mice (d–g) and 100 days after adoptive transfer of CD4 + cell from longtime surviving secondary CBA recipients with B6 beating heart (h). Fresh 4-μm-thick graft cryosections were incubated with anti-CD4, CD8, and CD68 monoclonal antibody or anti-Foxp3 polyclonal antibody. In (d–g), the left-hand panels show samples obtained from mice exercising on a treadmill, and the right-hand panels show samples from untreated mice (magnification ×40). In (h), all panels show samples obtained from longtime surviving transplant recipients in CD4 + cell adoptive transfer groups (magnification ×100) (i) CD4, CD25, and Foxp3 expression in splenocytes as determined by flow cytometry 1, 2, and 4 weeks after transplantation. The right-hand graph shows the percentage of CD4 + CD25 + Foxp3 + cells in the CD4 + cells as determined by flow cytometry. Data are mean ± SD values ( n = 5 mice in each group). MST median survival time. * P
Figure Legend Snippet: Evidence of generation of regulatory cells in treadmill-exercised CBA allograft recipients. (a) Scheme on adoptive transfer study to confirm the generation of regulatory T cells. (b, c) Cardiac allograft survival after adoptive transfer of whole splenocytes (b) or CD4 + cells (c). (d–h) Results of double immunostaining of cardiac allografts obtained 4 weeks after transplantation from untreated mice and postoperative 1-week treadmill-exercised mice (d–g) and 100 days after adoptive transfer of CD4 + cell from longtime surviving secondary CBA recipients with B6 beating heart (h). Fresh 4-μm-thick graft cryosections were incubated with anti-CD4, CD8, and CD68 monoclonal antibody or anti-Foxp3 polyclonal antibody. In (d–g), the left-hand panels show samples obtained from mice exercising on a treadmill, and the right-hand panels show samples from untreated mice (magnification ×40). In (h), all panels show samples obtained from longtime surviving transplant recipients in CD4 + cell adoptive transfer groups (magnification ×100) (i) CD4, CD25, and Foxp3 expression in splenocytes as determined by flow cytometry 1, 2, and 4 weeks after transplantation. The right-hand graph shows the percentage of CD4 + CD25 + Foxp3 + cells in the CD4 + cells as determined by flow cytometry. Data are mean ± SD values ( n = 5 mice in each group). MST median survival time. * P

Techniques Used: Crocin Bleaching Assay, Adoptive Transfer Assay, Double Immunostaining, Transplantation Assay, Mouse Assay, Incubation, Expressing, Flow Cytometry, Cytometry, Microscale Thermophoresis

12) Product Images from "Relay of Herpes Simplex Virus between Langerhans Cells and Dermal Dendritic Cells in Human Skin"

Article Title: Relay of Herpes Simplex Virus between Langerhans Cells and Dermal Dendritic Cells in Human Skin

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1004812

Interaction of HSV-1 infected LCs with DC-SIGN + dermal cells (dermal DCs/macrophages). (A) (B) (C) Foreskin explants, 48 hr p.i. (A) LCs and DC-SIGN + dermal cells interacted in clusters. (B) Proportion of clusters GFP + LC/DC-SIGN + dermal cells containing > 10 cells, n = 3, mean ± SEM, p*** > 0.001. (C) DC-SIGN + dermal cells with or without GFP expression were quantified in 20 representative fields per sample at 60x magnification from 3 separate samples. Mean ± SEM, p*** > 0.001 (D) Primary penile herpetic lesion, blue: DAPI, orange: langerin, red: DC-SIGN, green: gD1. E: epidermis, D: dermis. The dotted line represents the basement membrane. Maximum projections of Z-series are presented (A D). Scale bar in (A) indicates 15 μm and scale bars in (D) indicate 50 μm (white) and 15 μm (yellow), respectively.
Figure Legend Snippet: Interaction of HSV-1 infected LCs with DC-SIGN + dermal cells (dermal DCs/macrophages). (A) (B) (C) Foreskin explants, 48 hr p.i. (A) LCs and DC-SIGN + dermal cells interacted in clusters. (B) Proportion of clusters GFP + LC/DC-SIGN + dermal cells containing > 10 cells, n = 3, mean ± SEM, p*** > 0.001. (C) DC-SIGN + dermal cells with or without GFP expression were quantified in 20 representative fields per sample at 60x magnification from 3 separate samples. Mean ± SEM, p*** > 0.001 (D) Primary penile herpetic lesion, blue: DAPI, orange: langerin, red: DC-SIGN, green: gD1. E: epidermis, D: dermis. The dotted line represents the basement membrane. Maximum projections of Z-series are presented (A D). Scale bar in (A) indicates 15 μm and scale bars in (D) indicate 50 μm (white) and 15 μm (yellow), respectively.

Techniques Used: Infection, Expressing

Emigration of DCs from inner foreskin explants with or without HSV infection. Inner foreskin explants were cultured for 72 hr in 24 transwell plates (membrane with 5 μm diameter pore-size) with or without v-UL37GFP. Tissues were placed in the upper chamber and allogeneic PBMCs were placed in the bottom chamber as shown in Fig 1B . After 72 hr, cells were collected from bottom chambers and labelled for flow cytometry. (A) Cells were gated on viable cells (PI - ) then CD45 + HLA-DR + cells before identifying different subsets of DCs migrated out of tissues to the bottom chambers. (B) The percentage of viable CD45 + HLA-DR + cells co-expressing langerin and BDCA3 or DC-SIGN and the percentage of individual emigrated cells in the bottom wells of mock or infected samples are shown. Repeated measures ANOVA was used to test statistical significance, n = 3, mean ± SEM, p*
Figure Legend Snippet: Emigration of DCs from inner foreskin explants with or without HSV infection. Inner foreskin explants were cultured for 72 hr in 24 transwell plates (membrane with 5 μm diameter pore-size) with or without v-UL37GFP. Tissues were placed in the upper chamber and allogeneic PBMCs were placed in the bottom chamber as shown in Fig 1B . After 72 hr, cells were collected from bottom chambers and labelled for flow cytometry. (A) Cells were gated on viable cells (PI - ) then CD45 + HLA-DR + cells before identifying different subsets of DCs migrated out of tissues to the bottom chambers. (B) The percentage of viable CD45 + HLA-DR + cells co-expressing langerin and BDCA3 or DC-SIGN and the percentage of individual emigrated cells in the bottom wells of mock or infected samples are shown. Repeated measures ANOVA was used to test statistical significance, n = 3, mean ± SEM, p*

Techniques Used: Infection, Cell Culture, Flow Cytometry, Cytometry, Expressing

Migration of HSV infected LCs into the dermis and their subsequent interaction with BDCA3 + dermal DCs in clusters. (A) (B) (C) (D) LCs and BDCA3+ dDCs in the dermis of foreskin explants at 24 hr p.i. (A) 48 hr p.i. (B, C, D), yellow arrows: GFP + langerin + cells, green box: GFP + langerin + cells, yellow box: GFP + BDCA3 + cells, pink box: GFP/langerin + BDCA3 + dermal DCs. (C) Proportion of GFP + LC/BDCA3 + DC clusters with > 10 cells, n = 3, mean ± SEM, p***
Figure Legend Snippet: Migration of HSV infected LCs into the dermis and their subsequent interaction with BDCA3 + dermal DCs in clusters. (A) (B) (C) (D) LCs and BDCA3+ dDCs in the dermis of foreskin explants at 24 hr p.i. (A) 48 hr p.i. (B, C, D), yellow arrows: GFP + langerin + cells, green box: GFP + langerin + cells, yellow box: GFP + BDCA3 + cells, pink box: GFP/langerin + BDCA3 + dermal DCs. (C) Proportion of GFP + LC/BDCA3 + DC clusters with > 10 cells, n = 3, mean ± SEM, p***

Techniques Used: Migration, Infection

Schemes of experimental systems used to examine LCs and dermal DC interaction (A) Serial sections of a primary herpetic lesion stained by H E (upper panels) and IF (lower panels). Blue: DAPI, green: HSV-1 glycoprotein D, orange: langerin, red: DC-SIGN. E: epidermis, D: dermis, S: section. Dotted line indicates basement membrane. Scale bar indicates 50 μm. (B) Scheme showing infection and processing the ex vivo inner foreskin tissue explant culture. (C) Scheme showing isolation of epidermal LCs and dermal DCs from abdominal skin.
Figure Legend Snippet: Schemes of experimental systems used to examine LCs and dermal DC interaction (A) Serial sections of a primary herpetic lesion stained by H E (upper panels) and IF (lower panels). Blue: DAPI, green: HSV-1 glycoprotein D, orange: langerin, red: DC-SIGN. E: epidermis, D: dermis, S: section. Dotted line indicates basement membrane. Scale bar indicates 50 μm. (B) Scheme showing infection and processing the ex vivo inner foreskin tissue explant culture. (C) Scheme showing isolation of epidermal LCs and dermal DCs from abdominal skin.

Techniques Used: Staining, Infection, Ex Vivo, Isolation

13) Product Images from "Combining a CD20 Chimeric Antigen Receptor and an Inducible Caspase 9 Suicide Switch to Improve the Efficacy and Safety of T Cell Adoptive Immunotherapy for Lymphoma"

Article Title: Combining a CD20 Chimeric Antigen Receptor and an Inducible Caspase 9 Suicide Switch to Improve the Efficacy and Safety of T Cell Adoptive Immunotherapy for Lymphoma

Journal: PLoS ONE

doi: 10.1371/journal.pone.0082742

CD20-specific effector activity of transduced T cells in vitro. ( A ) Transduced T cells exhibit CD20-specific cytolytic activity in vitro. iC9-CD20CAR-Δ19 T cells transduced with CD20-CAR (CD20-CAR T), or non-transduced (mock) T cells were co-cultured with EL4, Daudi, and Granta in various effector to target (E:T) ratios. Mean percent killing (± SD of triplicate culture) was determined by 4-hour standard 51 C release assay. ( B ) Anti-tumor effect of the transduced T cells. Mock or iC9-CD20CAR-Δ19 transduced T cells were co-cultured with CD20 + Ramos cells in various Effector to Target ratios (E:T) for 48 hours. Cells were then stained with antibodies recognizing CD22 and CD3. Flow cytometric analysis was used to determine the presence of Ramos cells (CD22 + CD3 − ) and T cells (CD22 − CD3 + ). Similar results were obtained with three independent experiments. ( C ) Cytokine production. Expanded T cells secreted IL2, IFN-γ and TNF-α after co-culture with CD20-expressing Ramos target cells for 48 hours (mean ± SD of triplicate samples). There were negligible levels of cytokine production when the expanded T cells were cultured alone with no target cells. Results are representative of three independent experiments.
Figure Legend Snippet: CD20-specific effector activity of transduced T cells in vitro. ( A ) Transduced T cells exhibit CD20-specific cytolytic activity in vitro. iC9-CD20CAR-Δ19 T cells transduced with CD20-CAR (CD20-CAR T), or non-transduced (mock) T cells were co-cultured with EL4, Daudi, and Granta in various effector to target (E:T) ratios. Mean percent killing (± SD of triplicate culture) was determined by 4-hour standard 51 C release assay. ( B ) Anti-tumor effect of the transduced T cells. Mock or iC9-CD20CAR-Δ19 transduced T cells were co-cultured with CD20 + Ramos cells in various Effector to Target ratios (E:T) for 48 hours. Cells were then stained with antibodies recognizing CD22 and CD3. Flow cytometric analysis was used to determine the presence of Ramos cells (CD22 + CD3 − ) and T cells (CD22 − CD3 + ). Similar results were obtained with three independent experiments. ( C ) Cytokine production. Expanded T cells secreted IL2, IFN-γ and TNF-α after co-culture with CD20-expressing Ramos target cells for 48 hours (mean ± SD of triplicate samples). There were negligible levels of cytokine production when the expanded T cells were cultured alone with no target cells. Results are representative of three independent experiments.

Techniques Used: Activity Assay, In Vitro, Transduction, Cell Culture, Release Assay, Staining, Flow Cytometry, Co-Culture Assay, Expressing

Expansion of transduced T cells using the NIH3T3-based AAPCs. ( A ) Rapid expansion of transduced T cells. Eight days after activation with CD3/28 beads, transduced T cells were cultured in plates coated with irradiated NIH3T3-20/80 (broken line) cells or NIH3T3-IV cells (solid line) in the presence of IL-2 (20 U/ml) and IL-15 (10 ng/ml on day 1 and 1 ng/ml subsequently). Cell counts were enumerated every 7 days by trypan blue exclusion. Absolute numbers of cells at various time points are depicted as mean ± SD of triplicate wells. Results are representative of three independent experiments. ( B ) Preferential enrichment of transduced T cells when co-cultured with 3T3-derived AAPCs. Eight days after activation with CD3/28 beads, transduced T cells were cultured in plates coated with irradiated NIH3T3-20/80 cells or NIH3T3-IV cells. The percentage of CD19 + cells was determined every 7 days by flow cytometirc analysis using a PE-conjugated mouse anti-human CD19 antibody. Results are shown as the mean percentage of CD19 + cells ± SD of triplicate wells at various time points and are representative of three independent experiments. ( C ) Central Memory Phenotype of expanded T cells (CD45RO + CD28 + CD62L + ). Flow cytometric analysis of T cells expanded after 2 re-stimulation cycles using NIH3T3 AAPCs was performed to determine the surface immunophenotype. The percent positivity (mean ± SD of triplicate cultures) for CD28, CD62L, CD45RO, and CD45RA was determined by corresponding antibody staining. Cells were gated on CD3 + CD19 + T cells. Similar results were obtained with 4 independent experiments using cells cultured after either 2 or 3 re-stimulation cycles. Results were concordant in 5 experiments.
Figure Legend Snippet: Expansion of transduced T cells using the NIH3T3-based AAPCs. ( A ) Rapid expansion of transduced T cells. Eight days after activation with CD3/28 beads, transduced T cells were cultured in plates coated with irradiated NIH3T3-20/80 (broken line) cells or NIH3T3-IV cells (solid line) in the presence of IL-2 (20 U/ml) and IL-15 (10 ng/ml on day 1 and 1 ng/ml subsequently). Cell counts were enumerated every 7 days by trypan blue exclusion. Absolute numbers of cells at various time points are depicted as mean ± SD of triplicate wells. Results are representative of three independent experiments. ( B ) Preferential enrichment of transduced T cells when co-cultured with 3T3-derived AAPCs. Eight days after activation with CD3/28 beads, transduced T cells were cultured in plates coated with irradiated NIH3T3-20/80 cells or NIH3T3-IV cells. The percentage of CD19 + cells was determined every 7 days by flow cytometirc analysis using a PE-conjugated mouse anti-human CD19 antibody. Results are shown as the mean percentage of CD19 + cells ± SD of triplicate wells at various time points and are representative of three independent experiments. ( C ) Central Memory Phenotype of expanded T cells (CD45RO + CD28 + CD62L + ). Flow cytometric analysis of T cells expanded after 2 re-stimulation cycles using NIH3T3 AAPCs was performed to determine the surface immunophenotype. The percent positivity (mean ± SD of triplicate cultures) for CD28, CD62L, CD45RO, and CD45RA was determined by corresponding antibody staining. Cells were gated on CD3 + CD19 + T cells. Similar results were obtained with 4 independent experiments using cells cultured after either 2 or 3 re-stimulation cycles. Results were concordant in 5 experiments.

Techniques Used: Activation Assay, Cell Culture, Irradiation, Derivative Assay, Flow Cytometry, Staining

14) Product Images from "The 20S proteasome activator PA28γ controls the compaction of chromatin"

Article Title: The 20S proteasome activator PA28γ controls the compaction of chromatin

Journal: bioRxiv

doi: 10.1101/716332

PA28γ depletion decreases the S phase duration. A. Asynchronous parental (WT) and KO-PA28γ U2OS cells were fixed and stained with propidium iodide and then subjected to flow cytometry analysis. The histogram presents the repartition of the cells in the G1, S, and G2/M phases of the cell cycle. Data represent the means ± SD from four independent biological repeats. p -values were determined by 2-way ANOVA test, ns = not significant ( p = 0.2407), * p
Figure Legend Snippet: PA28γ depletion decreases the S phase duration. A. Asynchronous parental (WT) and KO-PA28γ U2OS cells were fixed and stained with propidium iodide and then subjected to flow cytometry analysis. The histogram presents the repartition of the cells in the G1, S, and G2/M phases of the cell cycle. Data represent the means ± SD from four independent biological repeats. p -values were determined by 2-way ANOVA test, ns = not significant ( p = 0.2407), * p

Techniques Used: Staining, Flow Cytometry

Endogenous HP1β, PA28 and the 20S proteasome co-localize in U2OS cells. A. Nuclear localization of endogenous HP1β (left panel) and PA28γ (middle panel) by immunofluorescence in asynchronous U2OS cells after pre-permeabilization with 0.5% Triton X-100. A representative merged image of HP1β (green) and PA28γ (red), and higher-magnification views are shown (right panel). Scale bars, 10 μm. B. A representative Airyscan confocal Z-projected image showing the co-detection of HP1β (green) and PA28γ (red) (left). Co-localizations of both proteins along the cross is shown. Scale bars, 5 μm. Using the co-localization module of Imaris, a representative image of HP1β (green), PA28γ (red) and co-localization spots (white/grey) corresponding to 3-D image (middle panel) is shown with the corresponding image showing only co-localization spots (right panel). Scale bars, 5 μm. C. Immunoblot analysis of PA28γ expression level in total extracts of U2OS cells treated or not with si-PA28γ, used for in situ Proximity Ligation Assay ( is -PLA) (left panel). Tubulin was used as a loading control. The relative abundance of PA28γ was quantified using ImageJ software. Control (CTL) or si-PA28γ treated U2OS cells were subjected to is -PLA using primary antibodies directed against HP1 β and PA28γ, and DNA stained with DAPI. Positive PLA signals appear as green dots and higher magnification views of a nucleus are shown (middle panel). Scale bars, 10 μm. Quantification of PLA dots was carried out using an ImageJ plugin (see Materials and Methods). The number of PLA dots per nucleus for HP1β/PA28γ interaction in control (CTL) or si-PA28γ treated cells is shown graphically (right panel). Data represent the means ± SD from 3 independent experiments, the number of analyzed cells is n = 78 and n = 45 in control and si-PA28γ treated cells, respectively. The p -value was determined with Student’s T-test, **** ( p = 0.0001). D. Immunoblot analysis of HP1β expression level in total extracts from U2OS cells treated or not with si-HP1β (upper left panel). Tubulin was used as a loading control. The relative abundance of HP1β proteins was quantified using ImageJ software. Control (CTL) or si-HP1β treated U2OS cells were subjected to is -PLA using primary antibodies directed against HP1β and α4 (a subunit of the 20S proteasome), and DNA was stained with DAPI. Positive PLA signals appear as green dots. A higher magnification view of a nucleus is shown (lower left panel). Scale bar, 10 μm. The number of PLA dots per nucleus for HP1β/α4 interaction in control (CTL) or si-HP1β treated cells is shown on the bar graph (right panel). Data represent the means ± SD from 3 independent experiments, the number of cells analyzed is n = 48 and n = 46 in control cells and si-HP1β treated cells, respectively. p -value was determined with Student’s T-test, ****( p
Figure Legend Snippet: Endogenous HP1β, PA28 and the 20S proteasome co-localize in U2OS cells. A. Nuclear localization of endogenous HP1β (left panel) and PA28γ (middle panel) by immunofluorescence in asynchronous U2OS cells after pre-permeabilization with 0.5% Triton X-100. A representative merged image of HP1β (green) and PA28γ (red), and higher-magnification views are shown (right panel). Scale bars, 10 μm. B. A representative Airyscan confocal Z-projected image showing the co-detection of HP1β (green) and PA28γ (red) (left). Co-localizations of both proteins along the cross is shown. Scale bars, 5 μm. Using the co-localization module of Imaris, a representative image of HP1β (green), PA28γ (red) and co-localization spots (white/grey) corresponding to 3-D image (middle panel) is shown with the corresponding image showing only co-localization spots (right panel). Scale bars, 5 μm. C. Immunoblot analysis of PA28γ expression level in total extracts of U2OS cells treated or not with si-PA28γ, used for in situ Proximity Ligation Assay ( is -PLA) (left panel). Tubulin was used as a loading control. The relative abundance of PA28γ was quantified using ImageJ software. Control (CTL) or si-PA28γ treated U2OS cells were subjected to is -PLA using primary antibodies directed against HP1 β and PA28γ, and DNA stained with DAPI. Positive PLA signals appear as green dots and higher magnification views of a nucleus are shown (middle panel). Scale bars, 10 μm. Quantification of PLA dots was carried out using an ImageJ plugin (see Materials and Methods). The number of PLA dots per nucleus for HP1β/PA28γ interaction in control (CTL) or si-PA28γ treated cells is shown graphically (right panel). Data represent the means ± SD from 3 independent experiments, the number of analyzed cells is n = 78 and n = 45 in control and si-PA28γ treated cells, respectively. The p -value was determined with Student’s T-test, **** ( p = 0.0001). D. Immunoblot analysis of HP1β expression level in total extracts from U2OS cells treated or not with si-HP1β (upper left panel). Tubulin was used as a loading control. The relative abundance of HP1β proteins was quantified using ImageJ software. Control (CTL) or si-HP1β treated U2OS cells were subjected to is -PLA using primary antibodies directed against HP1β and α4 (a subunit of the 20S proteasome), and DNA was stained with DAPI. Positive PLA signals appear as green dots. A higher magnification view of a nucleus is shown (lower left panel). Scale bar, 10 μm. The number of PLA dots per nucleus for HP1β/α4 interaction in control (CTL) or si-HP1β treated cells is shown on the bar graph (right panel). Data represent the means ± SD from 3 independent experiments, the number of cells analyzed is n = 48 and n = 46 in control cells and si-HP1β treated cells, respectively. p -value was determined with Student’s T-test, ****( p

Techniques Used: Immunofluorescence, Expressing, In Situ, Proximity Ligation Assay, Software, Staining

PA28γ -depletion does not alter the expression level of H2B-GFP or mCherry-H2B, and analysis of re-expression of WT- and Δ C-PA28γ in HeLa H2B-2FPs cells. A. Immunoblot analysis of H2B-GFP and mCherry-H2B expression level in total extracts from parental (WT) and KO-PA28γ HeLa H2B-2FPs cells (left panel). Tubulin was used as a loading control. The relative abundance of HP1 β proteins was quantified using ImageJ software. Graphical representation of the relative abundance of H2B-GFP and mCherry-H2B normalized to tubulin (right panel). The mean ± SD is from four independent experiments. Statistical significance was evaluated based on Student’s T-test, ns = not significant. ( p = 0.2027 and 0.4024 for H2B-GFP and mCherry-H2B, respectively) B. Quantification of the H2B-GFP and mCherry-H2B fluorescence intensities in WT and KO-PA28γ HeLa H2B-2FPs cells. The total number of analyzed cells is n = 172 (WT), n = 183 (KO-PA28γ). Statistical significance was evaluated with Student’s T-test, ns= not significant. C. FRET analysis in WT, KO-PA28γ HeLa H2B-FPs cells, and WT HeLa H2B-FPs cells treated with Trichostatin A (TSA, 200ng/ml, 24h). The statistical analysis of the mean FRET efficiency percentage is presented as box-and-whisker plots. The thick line represents median, the boxes correspond to the mean FRET values upper and lower of the median, with the whiskers covering the 10-90 percentile range. The total number of analyzed nuclei is n = 154 (WT), n = 132 (KO-PA28γ), and n = 33 (WT + TSA), **** p
Figure Legend Snippet: PA28γ -depletion does not alter the expression level of H2B-GFP or mCherry-H2B, and analysis of re-expression of WT- and Δ C-PA28γ in HeLa H2B-2FPs cells. A. Immunoblot analysis of H2B-GFP and mCherry-H2B expression level in total extracts from parental (WT) and KO-PA28γ HeLa H2B-2FPs cells (left panel). Tubulin was used as a loading control. The relative abundance of HP1 β proteins was quantified using ImageJ software. Graphical representation of the relative abundance of H2B-GFP and mCherry-H2B normalized to tubulin (right panel). The mean ± SD is from four independent experiments. Statistical significance was evaluated based on Student’s T-test, ns = not significant. ( p = 0.2027 and 0.4024 for H2B-GFP and mCherry-H2B, respectively) B. Quantification of the H2B-GFP and mCherry-H2B fluorescence intensities in WT and KO-PA28γ HeLa H2B-2FPs cells. The total number of analyzed cells is n = 172 (WT), n = 183 (KO-PA28γ). Statistical significance was evaluated with Student’s T-test, ns= not significant. C. FRET analysis in WT, KO-PA28γ HeLa H2B-FPs cells, and WT HeLa H2B-FPs cells treated with Trichostatin A (TSA, 200ng/ml, 24h). The statistical analysis of the mean FRET efficiency percentage is presented as box-and-whisker plots. The thick line represents median, the boxes correspond to the mean FRET values upper and lower of the median, with the whiskers covering the 10-90 percentile range. The total number of analyzed nuclei is n = 154 (WT), n = 132 (KO-PA28γ), and n = 33 (WT + TSA), **** p

Techniques Used: Expressing, Software, Fluorescence, Whisker Assay

PA28γ is present on heterochromatin and its depletion induces a decompaction of pericentromeric heterochromatin. A. ChIP-qPCR analysis of PA28γ levels at different repetitive elements located in heterochromatin or euchromatin (as indicated on the x-axis) in wild type (WT) versus KO-PA28γ U2OS cells (right panel). Data are represented as relative enrichment of PA28γ antibody versus H3 control as shown on the y-axis. Data are means ± SEM ( n = 5). Significance was calculated by Student’s T-test, ** p
Figure Legend Snippet: PA28γ is present on heterochromatin and its depletion induces a decompaction of pericentromeric heterochromatin. A. ChIP-qPCR analysis of PA28γ levels at different repetitive elements located in heterochromatin or euchromatin (as indicated on the x-axis) in wild type (WT) versus KO-PA28γ U2OS cells (right panel). Data are represented as relative enrichment of PA28γ antibody versus H3 control as shown on the y-axis. Data are means ± SEM ( n = 5). Significance was calculated by Student’s T-test, ** p

Techniques Used: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

Co-localization of PA28γ with HP1α and HP1β /PA28γ co-localization is independent of PIP30, a regulator of PA28γ. A. Immunoblot of whole cell extract (30 μg) from asynchronous parental (WT) and KO-PA28γ (KO-PA28γ) U2OS cells, using anti-PA28γ. Tubulin was used as a loading control (left panel). Asynchronously-growing U2OS-KO-PA28γ cells were pre-permeabilized with 0.5 % Triton-X100 before fixation and the detection of endogenous HP1β (left panel) and PA28γ (middle panel) by indirect immunofluorescence using anti-HP1β and PA28γ antibodies. A representative merged image of HP1β (green) and PA28γ (red) is shown (right panel). Scale bars, 10 μm. B. In situ Proximity ligation assay ( is -PLA) was carried out in asynchronous U2OS cell line. Fixed cells were treated with primary antibodies directed against PA28γ (mouse monoclonal) and HP1α (rabbit polyclonal) (CTL) or with only PA28γ antibodies (w/o anti-HP1α) and DNA was stained with DAPI (left panel). A higher magnification view of a nucleus is shown. Scale bars, 10 μm. The number of PLA dots per nucleus in cells treated with both antibodies (CTL) or with only PA28γ antibodies (w/o anti-HP1α) is shown on the bar graph (right panel). Data represent the mean ± SD from 3 independent experiments, the number of cells analyzed was n = 40 and n = 41 in control cells and cells treated without primary HP1 α antibody, respectively. The p -value was determined with Student’s T-test, **** ( p ≤ 0.0001). C. Whole-cell extracts (30 μg) of parental (WT), PA28γ-knockout (KO-PA28γ) and PIP30-knock-out (KO-PIP30) U2OS cells used for the is -PLA were analyzed by SDS-PAGE and immunoblotted with the antibodies indicated (left panel). Is -PLA was carried out using primary antibodies directed against HP1β and PA28γ, and DNA was stained with DAPI. Representative images of parental (WT) and KO-PIP30 U2OS cells are presented and higher magnification views are shown (middle panel). Scale bars, 10 μm. Quantification of PLA-dots was performed as in Fig 2C . The number of PLA-dots per nucleus is shown on the bar graph (right panel). Data represent the mean ± SD from 3 independent experiments; the number of cells analyzed was n = 59 (WT), n = 40 (KO-PIP30) and n = 42 (KO-PA28γ). Statistical significance was evaluated based on one-way ANOVA analysis (ns = not significant, and *** p
Figure Legend Snippet: Co-localization of PA28γ with HP1α and HP1β /PA28γ co-localization is independent of PIP30, a regulator of PA28γ. A. Immunoblot of whole cell extract (30 μg) from asynchronous parental (WT) and KO-PA28γ (KO-PA28γ) U2OS cells, using anti-PA28γ. Tubulin was used as a loading control (left panel). Asynchronously-growing U2OS-KO-PA28γ cells were pre-permeabilized with 0.5 % Triton-X100 before fixation and the detection of endogenous HP1β (left panel) and PA28γ (middle panel) by indirect immunofluorescence using anti-HP1β and PA28γ antibodies. A representative merged image of HP1β (green) and PA28γ (red) is shown (right panel). Scale bars, 10 μm. B. In situ Proximity ligation assay ( is -PLA) was carried out in asynchronous U2OS cell line. Fixed cells were treated with primary antibodies directed against PA28γ (mouse monoclonal) and HP1α (rabbit polyclonal) (CTL) or with only PA28γ antibodies (w/o anti-HP1α) and DNA was stained with DAPI (left panel). A higher magnification view of a nucleus is shown. Scale bars, 10 μm. The number of PLA dots per nucleus in cells treated with both antibodies (CTL) or with only PA28γ antibodies (w/o anti-HP1α) is shown on the bar graph (right panel). Data represent the mean ± SD from 3 independent experiments, the number of cells analyzed was n = 40 and n = 41 in control cells and cells treated without primary HP1 α antibody, respectively. The p -value was determined with Student’s T-test, **** ( p ≤ 0.0001). C. Whole-cell extracts (30 μg) of parental (WT), PA28γ-knockout (KO-PA28γ) and PIP30-knock-out (KO-PIP30) U2OS cells used for the is -PLA were analyzed by SDS-PAGE and immunoblotted with the antibodies indicated (left panel). Is -PLA was carried out using primary antibodies directed against HP1β and PA28γ, and DNA was stained with DAPI. Representative images of parental (WT) and KO-PIP30 U2OS cells are presented and higher magnification views are shown (middle panel). Scale bars, 10 μm. Quantification of PLA-dots was performed as in Fig 2C . The number of PLA-dots per nucleus is shown on the bar graph (right panel). Data represent the mean ± SD from 3 independent experiments; the number of cells analyzed was n = 59 (WT), n = 40 (KO-PIP30) and n = 42 (KO-PA28γ). Statistical significance was evaluated based on one-way ANOVA analysis (ns = not significant, and *** p

Techniques Used: Immunofluorescence, In Situ, Proximity Ligation Assay, Staining, Knock-Out, SDS Page

PA28γ controls chromatin compaction, independently of its interaction with the 20S proteasome. A. Immunoblot analysis of PA28γ expression level in total extracts from parental (WT) and PA28γ-knockout (KO-PA28γ) HeLa H2B-2FPs cells. Tubulin was used as a loading control. B. FRET analysis in asynchronous parental (WT) and PA28γ-knockout (KO-PA28γ) HeLa H2B-FPs cells. FLIM-FRET measurements were performed and the spatial distribution of the FRET efficiency is represented in a continuous pseudo-color scale ranging from 0 to 30 % (left panel). Scale bars, 10 μm. Right panel, statistical analysis of the mean FRET efficiency percentage in WT and KO-PA28γ HeLa H2B-2FPs nuclei, presented as box-and-whisker plots. The thick line represents median, the boxes correspond to the mean FRET values upper and lower of the median, with the whiskers covering the 10-90 percentile range. Data represent the means ± SD from 4-6 independent experiments, the total number of analyzed cells is n = 154 nuclei (WT) and n = 132 nuclei (KO-PA28γ), **** p
Figure Legend Snippet: PA28γ controls chromatin compaction, independently of its interaction with the 20S proteasome. A. Immunoblot analysis of PA28γ expression level in total extracts from parental (WT) and PA28γ-knockout (KO-PA28γ) HeLa H2B-2FPs cells. Tubulin was used as a loading control. B. FRET analysis in asynchronous parental (WT) and PA28γ-knockout (KO-PA28γ) HeLa H2B-FPs cells. FLIM-FRET measurements were performed and the spatial distribution of the FRET efficiency is represented in a continuous pseudo-color scale ranging from 0 to 30 % (left panel). Scale bars, 10 μm. Right panel, statistical analysis of the mean FRET efficiency percentage in WT and KO-PA28γ HeLa H2B-2FPs nuclei, presented as box-and-whisker plots. The thick line represents median, the boxes correspond to the mean FRET values upper and lower of the median, with the whiskers covering the 10-90 percentile range. Data represent the means ± SD from 4-6 independent experiments, the total number of analyzed cells is n = 154 nuclei (WT) and n = 132 nuclei (KO-PA28γ), **** p

Techniques Used: Expressing, Knock-Out, Whisker Assay

PA28γ and HP1β co-localize in α 4-GFP-20S proteasome foci. A. Stable asynchronously growing U2OS (Tet-Off) α4-GFP cells were induced for the expression of GFP-tagged α4 subunit of the 20S proteasome (α4-GFP, green) for 24 hours in the absence of tetracycline, then fixed and stained with DAPI (blue). Scale bar, 10 μm. B. Induced U2OS-α4-GFP cells (α4-GFP, green), as in A, were immunostained with antibody raised against alpha 6 subunit of the 20S proteasome (α6, left panel), the regulatory complex PA28γ (PA28γ, middle panel) and a subunit of the 19S regulatory complex (Rpt6, right panel), all in grey. Scale bar, 10 μm. C. α4-GFP foci are associated with chromatin. Induced U2OS-α4-GFP cells (α4-GFP, green), as in A, were not treated (- CSK buffer) or treated (+ CSK buffer) with an extraction CSK buffer, then fixed and immunostained with anti-PA28γ antibodies (red). Scale bar, 5 μm. D. Induced U2OS-α4-GFP cells (α4-GFP, green) were immunostained with anti-HP1β antibodies (grey). A merge image of GFP and HP1β signal is shown. Scale bar, 10 μm. Arrows indicate sites of co-localization between HP1β and α4-GFP. E. Co-immunoprecipitation of PA28γ and HP1β in asynchronous induced U2OS-α4-GFP cells. Induced U2OS-α4-GFP cells, as in A, were lysed and subjected to pull-down with either an antibody raised against PA28γ or GFP-TRAP, or the appropriate isotype control (CTL). An immunoblot of the pull-down (IP) and supernatant (SN) from whole-cell extracts (WCE) was probed with the antibodies indicated. F. U2OS-α4-GFP cells were transfected with si-Luc, si-PA28γ or si-HP1β. One day later, the expression of α4-GFP was induced, and cells were recovered 48 hours after siRNA treatment. Immunostaining was performed to detect PA28γ (red) and HP1β (magenta) in cells treated with the siRNA indicated. Representative images are shown (left). Arrows indicate cells with α4-GFP and PA28γ foci. Scale bar, 10 μm. The percentage of cells with α4-GFP foci is shown in the bar graph (right). Error bars derived from 3 independent experiments represent the mean ± SD, n ≥ 47 cells per condition. One-way ANOVA analysis, p = 0.0001 (****) for siRNA-PA28γ and -HP1β versus WT.
Figure Legend Snippet: PA28γ and HP1β co-localize in α 4-GFP-20S proteasome foci. A. Stable asynchronously growing U2OS (Tet-Off) α4-GFP cells were induced for the expression of GFP-tagged α4 subunit of the 20S proteasome (α4-GFP, green) for 24 hours in the absence of tetracycline, then fixed and stained with DAPI (blue). Scale bar, 10 μm. B. Induced U2OS-α4-GFP cells (α4-GFP, green), as in A, were immunostained with antibody raised against alpha 6 subunit of the 20S proteasome (α6, left panel), the regulatory complex PA28γ (PA28γ, middle panel) and a subunit of the 19S regulatory complex (Rpt6, right panel), all in grey. Scale bar, 10 μm. C. α4-GFP foci are associated with chromatin. Induced U2OS-α4-GFP cells (α4-GFP, green), as in A, were not treated (- CSK buffer) or treated (+ CSK buffer) with an extraction CSK buffer, then fixed and immunostained with anti-PA28γ antibodies (red). Scale bar, 5 μm. D. Induced U2OS-α4-GFP cells (α4-GFP, green) were immunostained with anti-HP1β antibodies (grey). A merge image of GFP and HP1β signal is shown. Scale bar, 10 μm. Arrows indicate sites of co-localization between HP1β and α4-GFP. E. Co-immunoprecipitation of PA28γ and HP1β in asynchronous induced U2OS-α4-GFP cells. Induced U2OS-α4-GFP cells, as in A, were lysed and subjected to pull-down with either an antibody raised against PA28γ or GFP-TRAP, or the appropriate isotype control (CTL). An immunoblot of the pull-down (IP) and supernatant (SN) from whole-cell extracts (WCE) was probed with the antibodies indicated. F. U2OS-α4-GFP cells were transfected with si-Luc, si-PA28γ or si-HP1β. One day later, the expression of α4-GFP was induced, and cells were recovered 48 hours after siRNA treatment. Immunostaining was performed to detect PA28γ (red) and HP1β (magenta) in cells treated with the siRNA indicated. Representative images are shown (left). Arrows indicate cells with α4-GFP and PA28γ foci. Scale bar, 10 μm. The percentage of cells with α4-GFP foci is shown in the bar graph (right). Error bars derived from 3 independent experiments represent the mean ± SD, n ≥ 47 cells per condition. One-way ANOVA analysis, p = 0.0001 (****) for siRNA-PA28γ and -HP1β versus WT.

Techniques Used: Expressing, Staining, Immunoprecipitation, Transfection, Immunostaining, Derivative Assay

HP1β is present at the same repetitive elements than PA28γ. Immunoblot analysis of HP1β expression level in total extracts from U2OS cells treated or not with si-HP1β (left panel). Tubulin was used as a loading control. The relative abundance of HP1β proteins was quantified using ImageJ software. ChIP-qPCR analysis of HP1β levels at different repetitive elements (as indicated on the x-axis) in U2OS cells treated with si-Luc or si-HP1β. Data are represented as relative enrichment of HP1β antibody versus H3 control as shown on the y-axis (right panel). Data are means +/- SEM ( n = 5). Significance was calculated with Student’s T-test, ns = not significant p ≥ 0.05, ** p
Figure Legend Snippet: HP1β is present at the same repetitive elements than PA28γ. Immunoblot analysis of HP1β expression level in total extracts from U2OS cells treated or not with si-HP1β (left panel). Tubulin was used as a loading control. The relative abundance of HP1β proteins was quantified using ImageJ software. ChIP-qPCR analysis of HP1β levels at different repetitive elements (as indicated on the x-axis) in U2OS cells treated with si-Luc or si-HP1β. Data are represented as relative enrichment of HP1β antibody versus H3 control as shown on the y-axis (right panel). Data are means +/- SEM ( n = 5). Significance was calculated with Student’s T-test, ns = not significant p ≥ 0.05, ** p

Techniques Used: Expressing, Software, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

PA28γ is a crucial factor for chromatin compaction. A. HeLa H2B-2FPs cells (WT) were transfected with control si-Luc, si-PA28γ, si-HP1β or a mix of both siRNAs (si-PA28γ/HP1β) for 48 hours. Immunoblot analysis of PA28γ and HP1β protein levels in HeLa 2FPs following siRNA treatments were performed. Tubulin and anti-β actin antibodies were used as loading controls. The relative abundance of PA28γ and HP1β proteins was quantified using ImageJ software. B. Quantification of the mean FRET efficiencies were presented as box-and-whisker plots where the thick line represents median, the boxes correspond to the mean FRET values upper and lower of the median, with the whiskers covering the 10-90 percentile range. Data represent the means ± SD from 4 independent experiments, the total number of analyzed cells is n = 152 (si-Luc), n = 85 (si-PA28γ), n = 73 (si-HP1β), n = 61 (si-PA28γ/HP1β). ns = not significant, *** p
Figure Legend Snippet: PA28γ is a crucial factor for chromatin compaction. A. HeLa H2B-2FPs cells (WT) were transfected with control si-Luc, si-PA28γ, si-HP1β or a mix of both siRNAs (si-PA28γ/HP1β) for 48 hours. Immunoblot analysis of PA28γ and HP1β protein levels in HeLa 2FPs following siRNA treatments were performed. Tubulin and anti-β actin antibodies were used as loading controls. The relative abundance of PA28γ and HP1β proteins was quantified using ImageJ software. B. Quantification of the mean FRET efficiencies were presented as box-and-whisker plots where the thick line represents median, the boxes correspond to the mean FRET values upper and lower of the median, with the whiskers covering the 10-90 percentile range. Data represent the means ± SD from 4 independent experiments, the total number of analyzed cells is n = 152 (si-Luc), n = 85 (si-PA28γ), n = 73 (si-HP1β), n = 61 (si-PA28γ/HP1β). ns = not significant, *** p

Techniques Used: Transfection, Software, Whisker Assay

Endogenous PA28γ and HP1β co-localize in HeLa cells. Immunoblot analysis of PA28γ expression level in total extracts from HeLa cells, treated or not with si-PA28γ (left panel). Tubulin was used as loading control (left panel). The relative abundance of PA28γ proteins was quantified using ImageJ software. Control (CTL) or si-PA28γ (si-PA28γ) treated HeLa cells were subjected to is -PLA using primary antibodies directed against HP1β and PA28γ, and DNA was stained with DAPI. Positive PLA signals appear as green dots, and a higher magnification view of a nucleus is shown (middle panel). Scale bars, 10 μm. Quantification of PLA dots was carried out using an ImageJ plugin (see Materials and Methods). The number of PLA dots per nucleus for HP1β/PA28γ interaction in control (CTL) or si-PA28γ treated cells is shown graphically (right panel). Data represent the means ± SD from 3 independent experiments, the number of analyzed cells is n = 40 and n = 34 in control and si-PA28γ treated cells, respectively. p value was determined with Student’s T-test, ** p = 0.0016.
Figure Legend Snippet: Endogenous PA28γ and HP1β co-localize in HeLa cells. Immunoblot analysis of PA28γ expression level in total extracts from HeLa cells, treated or not with si-PA28γ (left panel). Tubulin was used as loading control (left panel). The relative abundance of PA28γ proteins was quantified using ImageJ software. Control (CTL) or si-PA28γ (si-PA28γ) treated HeLa cells were subjected to is -PLA using primary antibodies directed against HP1β and PA28γ, and DNA was stained with DAPI. Positive PLA signals appear as green dots, and a higher magnification view of a nucleus is shown (middle panel). Scale bars, 10 μm. Quantification of PLA dots was carried out using an ImageJ plugin (see Materials and Methods). The number of PLA dots per nucleus for HP1β/PA28γ interaction in control (CTL) or si-PA28γ treated cells is shown graphically (right panel). Data represent the means ± SD from 3 independent experiments, the number of analyzed cells is n = 40 and n = 34 in control and si-PA28γ treated cells, respectively. p value was determined with Student’s T-test, ** p = 0.0016.

Techniques Used: Expressing, Software, Proximity Ligation Assay, Staining

PA28γ contributes to the maintenance of heterochromatin marks. A. Representative immunoblots of whole cell extracts from U2OS (WT and KO-PA28γ) cells, using anti-H3K9me3 antibodies. Histone H3 was used as loading control. Graphical representation of the relative abundance of the tri-methylation (H3K9me3) mark on histone H3 normalized to histone H3. The mean ± SD is from four independent experiments. The p - value was determined with a Student’s T-test, ns = not significant ( p = 0.9354). B. Immunoblots of whole cell extracts from U2OS (WT and KO-PA28γ) cells, using anti-H4K20me3 and anti-H4K20me1 antibodies. Histone H3 was used as loading control. Graphical representation of the relative abundance of the mono-methylation (H4K20me1) and the tri-methylation (H4K20me3) marks on histone H4 normalized to histone H3. The mean ± SD is from four independent experiments. The p -value was determined with a Student’s T-test, **** ( p ≤ 0.0001). C and D. ChIP-qPCR analysis of H3K9me3 and H4K20me3 levels in WT versus KO-PA28γ U2OS cells. Data are represented as relative enrichment of each specific antibody versus H3 control as shown on the y-axis. Data are means ± SEM ( n = 5 for H3K9me3 and H3, n = 3 for H4K20me3). Significance was calculated by Student’s T-test, ** p
Figure Legend Snippet: PA28γ contributes to the maintenance of heterochromatin marks. A. Representative immunoblots of whole cell extracts from U2OS (WT and KO-PA28γ) cells, using anti-H3K9me3 antibodies. Histone H3 was used as loading control. Graphical representation of the relative abundance of the tri-methylation (H3K9me3) mark on histone H3 normalized to histone H3. The mean ± SD is from four independent experiments. The p - value was determined with a Student’s T-test, ns = not significant ( p = 0.9354). B. Immunoblots of whole cell extracts from U2OS (WT and KO-PA28γ) cells, using anti-H4K20me3 and anti-H4K20me1 antibodies. Histone H3 was used as loading control. Graphical representation of the relative abundance of the mono-methylation (H4K20me1) and the tri-methylation (H4K20me3) marks on histone H4 normalized to histone H3. The mean ± SD is from four independent experiments. The p -value was determined with a Student’s T-test, **** ( p ≤ 0.0001). C and D. ChIP-qPCR analysis of H3K9me3 and H4K20me3 levels in WT versus KO-PA28γ U2OS cells. Data are represented as relative enrichment of each specific antibody versus H3 control as shown on the y-axis. Data are means ± SEM ( n = 5 for H3K9me3 and H3, n = 3 for H4K20me3). Significance was calculated by Student’s T-test, ** p

Techniques Used: Western Blot, Methylation, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

15) Product Images from "Calpain-2 regulates hypoxia/HIF-induced amoeboid reprogramming and metastasis"

Article Title: Calpain-2 regulates hypoxia/HIF-induced amoeboid reprogramming and metastasis

Journal: bioRxiv

doi: 10.1101/2020.01.06.892497

Reduced β1 integrin activity in amoeboid transition. a, In-silico modelling of cell elongation. Individual cell migration in dependence of friction and contractility using a two-dimensional phase field simulation. b, Active and total β1 integrin protein content (2D monolayer culture). D, densitometric analysis (representative Western blot, n=2-3). c, Schematic of cell isolation for harvesting attached (highly adhesive) and detached (weakly adhesive) cells in 2D culture. d, Active and total β1 integrin surface expression (MFI) in 4T1 subpopulations 48 h after treatment (left panel, representative flow cytometry histogram). Ratio of active/total β1 integrin surface expression normalized to vehicle control ratios (right panel). Columns show the median from independent experiments (data points). ** P=0.007, * P=0.03 (unpaired t-test, two-sided). e, Confocal micrographs of active (mAb 9EG7) and total β1 integrin (mAb CD29) expression of 4T1 tumoroids invading into 3D collagen (left panel) and the ratio of active/total β1 integrin per invading 4T1 single cell (right panel, 106 cells). Data show ratios of single cells; horizontal lines the median. Insets, single cell invasion phenotypes (arrowheads). Scale bars, 100 μm (overview), 10 µm (inset). **** P
Figure Legend Snippet: Reduced β1 integrin activity in amoeboid transition. a, In-silico modelling of cell elongation. Individual cell migration in dependence of friction and contractility using a two-dimensional phase field simulation. b, Active and total β1 integrin protein content (2D monolayer culture). D, densitometric analysis (representative Western blot, n=2-3). c, Schematic of cell isolation for harvesting attached (highly adhesive) and detached (weakly adhesive) cells in 2D culture. d, Active and total β1 integrin surface expression (MFI) in 4T1 subpopulations 48 h after treatment (left panel, representative flow cytometry histogram). Ratio of active/total β1 integrin surface expression normalized to vehicle control ratios (right panel). Columns show the median from independent experiments (data points). ** P=0.007, * P=0.03 (unpaired t-test, two-sided). e, Confocal micrographs of active (mAb 9EG7) and total β1 integrin (mAb CD29) expression of 4T1 tumoroids invading into 3D collagen (left panel) and the ratio of active/total β1 integrin per invading 4T1 single cell (right panel, 106 cells). Data show ratios of single cells; horizontal lines the median. Insets, single cell invasion phenotypes (arrowheads). Scale bars, 100 μm (overview), 10 µm (inset). **** P

Techniques Used: Activity Assay, In Silico, Migration, Western Blot, Cell Isolation, Expressing, Flow Cytometry

16) Product Images from "Batf is important for IL-4 expression in T follicular helper cells"

Article Title: Batf is important for IL-4 expression in T follicular helper cells

Journal: Nature Communications

doi: 10.1038/ncomms8997

Batf contributes to pro-allergic function of IL-4-expressing Tfh cells. ( a ) Male WT and Batf KO mice (6–8 weeks old, n =5 per group) were injected i.p. with 0.2 ml saline containing 100 μg Ova in Alum at 2 weeks interval. On day 14, mice were intranasally challenged with Ova followed by three more challenges at days 26, 27 and 28. Twenty-four hours after the last challenge mice were killed. Percentage of IL-4-expressing CD4 + CD44 hi CXCR5 hi PD1 hi (Tfh) cells and ratio of percentage of IL-4-expressing Tfh cells to percentage of total Tfh cells from spleen and lung lymph nodes of WT and Batf KO asthmatic mice were analysed by flow cytometry. ( b – f ) Bone marrow chimera mice were generated and subjected to asthma as described in the Methods section. WT (CD45.1 + ) and Batf KO (CD45.2 + ) Tfh cells from the asthmatic mice were sorted and transferred into male C57BL/6 (CD45.2 + ) or B6.SJL (CD45.1 + ; 6–8 weeks old, n =4) mice, respectively. Twenty-four hours later the mice were challenged with Ova intranasally for 5 days and analysed. ( b ) BALF was analysed to measure airway infiltrating cells. ( c ) Expression of indicated cytokines in the lung was analysed by qRT–PCR analysis. Data were normalized to beta-actin gene. ( d ) Levels of Ova-specific IgGs in the serum were analysed by ELISA. ( e ) Spleen cells from the recipient mice were restimulated with Ova for 72 h and effector Th2 cytokines were analysed by ELISA. ( f ) Lung and lung lymph node cells were stained with Pacific blue-labelled CD45.1 mAb, PerCP-labelled anti-CD4 mAb, FITC-labelled anti-CD44 mAb and biotinylated anti-CXCR5 mAb, followed by APC-labelled streptavidin (BD Biosciences). Donor WT (CD45.1 + ) and Batf KO (CD45.2 + ) CD4 + CD44 hi cells were assessed for CXRCR5 and IL-4 expression after restimulation with Ova for 24 h. Results shown are mean±s.e.m. and representative of at least two independent experiments. P values: *
Figure Legend Snippet: Batf contributes to pro-allergic function of IL-4-expressing Tfh cells. ( a ) Male WT and Batf KO mice (6–8 weeks old, n =5 per group) were injected i.p. with 0.2 ml saline containing 100 μg Ova in Alum at 2 weeks interval. On day 14, mice were intranasally challenged with Ova followed by three more challenges at days 26, 27 and 28. Twenty-four hours after the last challenge mice were killed. Percentage of IL-4-expressing CD4 + CD44 hi CXCR5 hi PD1 hi (Tfh) cells and ratio of percentage of IL-4-expressing Tfh cells to percentage of total Tfh cells from spleen and lung lymph nodes of WT and Batf KO asthmatic mice were analysed by flow cytometry. ( b – f ) Bone marrow chimera mice were generated and subjected to asthma as described in the Methods section. WT (CD45.1 + ) and Batf KO (CD45.2 + ) Tfh cells from the asthmatic mice were sorted and transferred into male C57BL/6 (CD45.2 + ) or B6.SJL (CD45.1 + ; 6–8 weeks old, n =4) mice, respectively. Twenty-four hours later the mice were challenged with Ova intranasally for 5 days and analysed. ( b ) BALF was analysed to measure airway infiltrating cells. ( c ) Expression of indicated cytokines in the lung was analysed by qRT–PCR analysis. Data were normalized to beta-actin gene. ( d ) Levels of Ova-specific IgGs in the serum were analysed by ELISA. ( e ) Spleen cells from the recipient mice were restimulated with Ova for 72 h and effector Th2 cytokines were analysed by ELISA. ( f ) Lung and lung lymph node cells were stained with Pacific blue-labelled CD45.1 mAb, PerCP-labelled anti-CD4 mAb, FITC-labelled anti-CD44 mAb and biotinylated anti-CXCR5 mAb, followed by APC-labelled streptavidin (BD Biosciences). Donor WT (CD45.1 + ) and Batf KO (CD45.2 + ) CD4 + CD44 hi cells were assessed for CXRCR5 and IL-4 expression after restimulation with Ova for 24 h. Results shown are mean±s.e.m. and representative of at least two independent experiments. P values: *

Techniques Used: Expressing, Mouse Assay, Injection, Flow Cytometry, Cytometry, Generated, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Staining

17) Product Images from "A B-Cell Superantigen Induces the Apoptosis of Murine and Human Malignant B Cells"

Article Title: A B-Cell Superantigen Induces the Apoptosis of Murine and Human Malignant B Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0162456

PpL induces increases of Bim and Bax and decreases of Bcl-2 proteins. Daudi cells were incubated in the presence of PpL (100 μg/ml) or PBS for the indicated times. Total proteins were obtained and Western blots were performed. Membranes were sequentially blotted with anti-Bax (A), anti-Bim (B) , anti-Bcl-2 (C) , or anti-actin antibodies. Images of representative Western blots are shown. The optical density of Bax, Bim and Bcl-2 bands were quantified and normalized to actin. The relative levels are shown. Data are presented as the mean ± SEM of three independent experiments (* p
Figure Legend Snippet: PpL induces increases of Bim and Bax and decreases of Bcl-2 proteins. Daudi cells were incubated in the presence of PpL (100 μg/ml) or PBS for the indicated times. Total proteins were obtained and Western blots were performed. Membranes were sequentially blotted with anti-Bax (A), anti-Bim (B) , anti-Bcl-2 (C) , or anti-actin antibodies. Images of representative Western blots are shown. The optical density of Bax, Bim and Bcl-2 bands were quantified and normalized to actin. The relative levels are shown. Data are presented as the mean ± SEM of three independent experiments (* p

Techniques Used: Incubation, Western Blot

Effect of caspase-8 inhibitor on the PpL-induced translocation of Bax, Bim and Bcl-2 between the mitochondrial and cytosol compartments. Daudi cells pretreated with caspase-8 inhibitor were incubated with PpL (100 μg/ml), OVA (100 μg/ml) or PBS for 8 hs. Cell lysates containing cytosol and mitochondria fractions were prepared. Expression of Bax, Bim, Bcl-2 and Bid was analyzed by Western blotting. Membranes were sequentially blotted with anti-Bax (A) , anti-Bim (B) , anti-Bcl-2 (C) , anti-Bid (D) , anti-actin or anti III complex antibodies. Images of representative Western blots are shown. The optical density of Bax, Bim, Bcl-2 and Bid bands was quantified and normalized to actin or III complex. The relative levels are shown. Data are presented as the mean ± SEM of three independent experiments, (** p
Figure Legend Snippet: Effect of caspase-8 inhibitor on the PpL-induced translocation of Bax, Bim and Bcl-2 between the mitochondrial and cytosol compartments. Daudi cells pretreated with caspase-8 inhibitor were incubated with PpL (100 μg/ml), OVA (100 μg/ml) or PBS for 8 hs. Cell lysates containing cytosol and mitochondria fractions were prepared. Expression of Bax, Bim, Bcl-2 and Bid was analyzed by Western blotting. Membranes were sequentially blotted with anti-Bax (A) , anti-Bim (B) , anti-Bcl-2 (C) , anti-Bid (D) , anti-actin or anti III complex antibodies. Images of representative Western blots are shown. The optical density of Bax, Bim, Bcl-2 and Bid bands was quantified and normalized to actin or III complex. The relative levels are shown. Data are presented as the mean ± SEM of three independent experiments, (** p

Techniques Used: Translocation Assay, Incubation, Expressing, Western Blot

Effect of caspase-9 inhibitor on the PpL-induced translocation of Bax, Bim and Bcl-2 between the mitochondrial and cytosol compartments. Daudi cells pretreated with caspase-9 inhibitor were incubated with PpL (100 μg/ml), OVA (100 μg/ml) or PBS for 8 hs. Cell lysates containing cytosol and mitochondria fractions were prepared. Expression of Bax, Bim, Bcl-2 and Bid was analyzed by Western blotting. Membranes were sequentially blotted with anti-Bax (A) , anti-Bim (B), anti-Bcl-2 (C) , anti-Bid (D) , anti-actin or anti-III complex antibodies. Images of representative Western blots are shown. The optical density of Bax, Bim, Bcl-2 and Bid bands was quantified and normalized to actin or III complex. The relative levels are shown. Data are presented as the mean ± SEM of three independent experiments, (*** p
Figure Legend Snippet: Effect of caspase-9 inhibitor on the PpL-induced translocation of Bax, Bim and Bcl-2 between the mitochondrial and cytosol compartments. Daudi cells pretreated with caspase-9 inhibitor were incubated with PpL (100 μg/ml), OVA (100 μg/ml) or PBS for 8 hs. Cell lysates containing cytosol and mitochondria fractions were prepared. Expression of Bax, Bim, Bcl-2 and Bid was analyzed by Western blotting. Membranes were sequentially blotted with anti-Bax (A) , anti-Bim (B), anti-Bcl-2 (C) , anti-Bid (D) , anti-actin or anti-III complex antibodies. Images of representative Western blots are shown. The optical density of Bax, Bim, Bcl-2 and Bid bands was quantified and normalized to actin or III complex. The relative levels are shown. Data are presented as the mean ± SEM of three independent experiments, (*** p

Techniques Used: Translocation Assay, Incubation, Expressing, Western Blot

PpL induces increases in Bax but not in Bcl-2 or Fas mRNA. Daudi cells were treated with PpL (100 μg/ml), OVA (100 μg/ml) or PBS. RNA was extracted and cDNA was obtained by RT-PCR and analyzed by semi-quantitative PCR for Bax, Fas, Bcl-2 and actin. Image of a representative result is shown. The OD of the bands of Bax, Bcl-2 and Fas cDNA was quantified and normalized to actin. The relative levels are shown. Data are presented as the mean ± SEM of four independent experiments, (*** p
Figure Legend Snippet: PpL induces increases in Bax but not in Bcl-2 or Fas mRNA. Daudi cells were treated with PpL (100 μg/ml), OVA (100 μg/ml) or PBS. RNA was extracted and cDNA was obtained by RT-PCR and analyzed by semi-quantitative PCR for Bax, Fas, Bcl-2 and actin. Image of a representative result is shown. The OD of the bands of Bax, Bcl-2 and Fas cDNA was quantified and normalized to actin. The relative levels are shown. Data are presented as the mean ± SEM of four independent experiments, (*** p

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

Translocation of Bim and Bax to the mitochondria and of Bcl2 to the cytosol. Daudi cells were incubated in the presence of PpL (100 μg/ml) or PBS for 8 hs. Cell lysates containing cytosol and mitochondria were prepared. Expression of Bax, Bim, Bcl2 and Bid were analyzed by Western blotting. Membranes were sequentially blotted with anti-Bax (A), anti-Bim (B) , anti-Bcl-2 (C) , anti-Bid (D) , anti-actin or anti-III complex antibodies. Images of representative Western blots are shown. The optical density of Bax, Bim, Bcl-2 and Bid bands were quantified and normalized to actin or anti-III complex. The relative levels are shown. Data are presented as the mean ± SEM of three independent experiments (** p
Figure Legend Snippet: Translocation of Bim and Bax to the mitochondria and of Bcl2 to the cytosol. Daudi cells were incubated in the presence of PpL (100 μg/ml) or PBS for 8 hs. Cell lysates containing cytosol and mitochondria were prepared. Expression of Bax, Bim, Bcl2 and Bid were analyzed by Western blotting. Membranes were sequentially blotted with anti-Bax (A), anti-Bim (B) , anti-Bcl-2 (C) , anti-Bid (D) , anti-actin or anti-III complex antibodies. Images of representative Western blots are shown. The optical density of Bax, Bim, Bcl-2 and Bid bands were quantified and normalized to actin or anti-III complex. The relative levels are shown. Data are presented as the mean ± SEM of three independent experiments (** p

Techniques Used: Translocation Assay, Incubation, Expressing, Western Blot

18) Product Images from "Engagement of Cytotoxic T Lymphocyte-associated Antigen 4 (CTLA-4) Induces Transforming Growth Factor ? (TGF-?) Production by Murine CD4+ T Cells "

Article Title: Engagement of Cytotoxic T Lymphocyte-associated Antigen 4 (CTLA-4) Induces Transforming Growth Factor ? (TGF-?) Production by Murine CD4+ T Cells

Journal: The Journal of Experimental Medicine

doi:

Engagement of CTLA-4 with antibody and stimulation of TCR by antigenic peptide upregulates TGF-β secretion. CD4 + T cell line CW-SW-1 specific for OVA peptide 323–339 was restimulated with OVA antigen (100 μg/ml) in the presence of normal BALB/c splenic APCs in X-Vivo-20. Anti–CTLA-4 (40 μg/ml) or the isotypic control hamster IgG ( Ham-IgG Ctrl ; 40 μg/ml) was added into the indicated culture wells. The supernatants were collected 72 h later and TGF-β was determined by ELISA.
Figure Legend Snippet: Engagement of CTLA-4 with antibody and stimulation of TCR by antigenic peptide upregulates TGF-β secretion. CD4 + T cell line CW-SW-1 specific for OVA peptide 323–339 was restimulated with OVA antigen (100 μg/ml) in the presence of normal BALB/c splenic APCs in X-Vivo-20. Anti–CTLA-4 (40 μg/ml) or the isotypic control hamster IgG ( Ham-IgG Ctrl ; 40 μg/ml) was added into the indicated culture wells. The supernatants were collected 72 h later and TGF-β was determined by ELISA.

Techniques Used: Enzyme-linked Immunosorbent Assay

Defect of CTLA-4–induced suppression of CD4 + T cell proliferation in TGF-β1 −/− mice. Freshly purified CD4 + T cells from asymptomatic TGF-β1 −/− mice were stimulated with the modified antibody regimen as indicated. Anti-CD3: 0.5 μg/ml; anti-CD28: 0.2 μg/ml; anti– CTLA-4 and hamster IgG ( Ham-IgG ): 20 μg/ml. Goat anti–hamster IgG (20 μg/ml) was added to all wells. ( A ) Surface expression of CTLA-4 on treated and control TGF-β1 −/− CD4 + T cells by flow cytometry. Cells were harvested after 56 h of culture and stained with FITC–anti-CD4 and PE–anti-CTLA-4. CD4 + T cells were gated and CTLA-4 staining of treated ( top ) and control ( middle ) cells on FL-2 channels is displayed. Bottom , negative control antibody staining for PE-conjugated anti-CTLA-4. ( B ) T cell proliferation of treated and control TGF-β1 −/− T cells. Data are expressed as mean ± SD ( CPM ) of triplicate wells.
Figure Legend Snippet: Defect of CTLA-4–induced suppression of CD4 + T cell proliferation in TGF-β1 −/− mice. Freshly purified CD4 + T cells from asymptomatic TGF-β1 −/− mice were stimulated with the modified antibody regimen as indicated. Anti-CD3: 0.5 μg/ml; anti-CD28: 0.2 μg/ml; anti– CTLA-4 and hamster IgG ( Ham-IgG ): 20 μg/ml. Goat anti–hamster IgG (20 μg/ml) was added to all wells. ( A ) Surface expression of CTLA-4 on treated and control TGF-β1 −/− CD4 + T cells by flow cytometry. Cells were harvested after 56 h of culture and stained with FITC–anti-CD4 and PE–anti-CTLA-4. CD4 + T cells were gated and CTLA-4 staining of treated ( top ) and control ( middle ) cells on FL-2 channels is displayed. Bottom , negative control antibody staining for PE-conjugated anti-CTLA-4. ( B ) T cell proliferation of treated and control TGF-β1 −/− T cells. Data are expressed as mean ± SD ( CPM ) of triplicate wells.

Techniques Used: Mouse Assay, Purification, Modification, Expressing, Flow Cytometry, Cytometry, Staining, Negative Control

CTLA-4 dose-dependent induction of TGF-β. ( A ) CD4 + Th2, Th1, and Th0 cell clones specific for OVA peptide 323–339 were restimulated with the antibody regimen as described for Fig. 3 A . TGF-β was determined by ELISA. The data are expressed as mean ± SD of duplicate cultures. ( B ) The CD4 + T cell clone 1A11 (Th0, Table 1 ) was stimulated with immobilized anti-CD3 (1 μg/ml) and different concentrations of anti–CTLA-4 or isotypic hamster IgG antibodies for 72 h. Supernatant TGF-β was determined in duplicate by ELISA, and the variations of the means are
Figure Legend Snippet: CTLA-4 dose-dependent induction of TGF-β. ( A ) CD4 + Th2, Th1, and Th0 cell clones specific for OVA peptide 323–339 were restimulated with the antibody regimen as described for Fig. 3 A . TGF-β was determined by ELISA. The data are expressed as mean ± SD of duplicate cultures. ( B ) The CD4 + T cell clone 1A11 (Th0, Table 1 ) was stimulated with immobilized anti-CD3 (1 μg/ml) and different concentrations of anti–CTLA-4 or isotypic hamster IgG antibodies for 72 h. Supernatant TGF-β was determined in duplicate by ELISA, and the variations of the means are

Techniques Used: Clone Assay, Enzyme-linked Immunosorbent Assay

TGF-β is associated with inhibition of CD4 +  T cell activation by CTLA-4 cross-linking. CD4 +  T cells isolated from B6 spleens  were cultured with anti-CD3 (2 μg/ml), anti-CD28 (5 μg/ml), and anti– CTLA-4 (20 μg/ml) followed by goat anti–hamster IgG ( Ham-IgG ; 20  μg/ml). Anti–TGF-β mAb or isotypic control antibody (mouse IgG1)  was included from the beginning of the culture at the indicated concentrations. Cells were cultured for 72 h. Data are expressed as mean cpm ±  SD of triplicate wells for [ 3 H]thymidine incorporation. Data not shown in  the figure are cpm of T cells incubated with medium alone (176 ± 26).  The experiments were repeated three times with similar results.
Figure Legend Snippet: TGF-β is associated with inhibition of CD4 + T cell activation by CTLA-4 cross-linking. CD4 + T cells isolated from B6 spleens were cultured with anti-CD3 (2 μg/ml), anti-CD28 (5 μg/ml), and anti– CTLA-4 (20 μg/ml) followed by goat anti–hamster IgG ( Ham-IgG ; 20 μg/ml). Anti–TGF-β mAb or isotypic control antibody (mouse IgG1) was included from the beginning of the culture at the indicated concentrations. Cells were cultured for 72 h. Data are expressed as mean cpm ± SD of triplicate wells for [ 3 H]thymidine incorporation. Data not shown in the figure are cpm of T cells incubated with medium alone (176 ± 26). The experiments were repeated three times with similar results.

Techniques Used: Inhibition, Activation Assay, Isolation, Cell Culture, Incubation

Cross-linking of CTLA-4 inhibits cytokine production by  CD4 +  T cells. CD4 +  T cells isolated from spleens of B6 mice were cultured  in complete DMEM only ( Med ) or with the indicated antibodies: anti– CTLA-4 (20 μg/ml) or control hamster IgG ( Ctrl ; 20 μg/ml) in the absence or presence of anti-CD3 (2 μg/ml) and anti-CD28 (5 μg/ml). Goat  anti–hamster IgG (heavy and light chains) antibody was then added to all  the wells at 20 μg/ml. T cell proliferation ( A ) was expressed as mean ± SD  of triplicate wells for  3 H incorporation ( CPM ). Secretion of IL-2 ( B ),  IFN-γ ( C ), and IL-4 ( D ) by CD4 +  T cells is shown. Supernatants were collected at 48 h, and the cytokine levels were determined by ELISA. The values are expressed as mean ± SD of replicate wells of ELISA plates.
Figure Legend Snippet: Cross-linking of CTLA-4 inhibits cytokine production by CD4 + T cells. CD4 + T cells isolated from spleens of B6 mice were cultured in complete DMEM only ( Med ) or with the indicated antibodies: anti– CTLA-4 (20 μg/ml) or control hamster IgG ( Ctrl ; 20 μg/ml) in the absence or presence of anti-CD3 (2 μg/ml) and anti-CD28 (5 μg/ml). Goat anti–hamster IgG (heavy and light chains) antibody was then added to all the wells at 20 μg/ml. T cell proliferation ( A ) was expressed as mean ± SD of triplicate wells for 3 H incorporation ( CPM ). Secretion of IL-2 ( B ), IFN-γ ( C ), and IL-4 ( D ) by CD4 + T cells is shown. Supernatants were collected at 48 h, and the cytokine levels were determined by ELISA. The values are expressed as mean ± SD of replicate wells of ELISA plates.

Techniques Used: Isolation, Mouse Assay, Cell Culture, Enzyme-linked Immunosorbent Assay

19) Product Images from "Dysregulation of junctional adhesion molecule-A via p63/GATA-3 in head and neck squamous cell carcinoma"

Article Title: Dysregulation of junctional adhesion molecule-A via p63/GATA-3 in head and neck squamous cell carcinoma

Journal: Oncotarget

doi: 10.18632/oncotarget.8432

Images of H.E. and immunohistochemical staining (A) of JAM-A, β-catenin, p63, ΔNp63 and GATA-3 in HNSCC and dysplastic regions Bar: 100 μm. Western blotting ( B ) and immunocytochemical staining ( C ) for p63, ΔNp63, JAM-A and β-catenin and flow cytometry ( D ) for JAM-A in Detroit562 cells incubated under hypoxia (2% O 2 ). Bars: 20 μm. ( E ) Real-time PCR for JAM-A mRNA in Detroit562 cells incubated under hypoxia (2% O 2 ). Results are given as means ± SE. p *
Figure Legend Snippet: Images of H.E. and immunohistochemical staining (A) of JAM-A, β-catenin, p63, ΔNp63 and GATA-3 in HNSCC and dysplastic regions Bar: 100 μm. Western blotting ( B ) and immunocytochemical staining ( C ) for p63, ΔNp63, JAM-A and β-catenin and flow cytometry ( D ) for JAM-A in Detroit562 cells incubated under hypoxia (2% O 2 ). Bars: 20 μm. ( E ) Real-time PCR for JAM-A mRNA in Detroit562 cells incubated under hypoxia (2% O 2 ). Results are given as means ± SE. p *

Techniques Used: Immunohistochemistry, Staining, Western Blot, Flow Cytometry, Cytometry, Incubation, Real-time Polymerase Chain Reaction

( A ) Images of H.E. and immunocytochemical staining for CK7, p63, ΔNp63, GATA-3, JAM-A and β-catenin in primary cultured cancer cells derived from HNSCC tissue. Bar: 100 μm. Western blotting ( B ) for p63, ΔNp63, GATA-3, JAM-A, β-catenin, occludin and claudin-1, 4, 7 and flow cytometry ( C ) for JAM-A HNSCC in primary cultured cancer cells transfected with siRNAs of p63, ΔNp63, GATA-3 and JAM-A.
Figure Legend Snippet: ( A ) Images of H.E. and immunocytochemical staining for CK7, p63, ΔNp63, GATA-3, JAM-A and β-catenin in primary cultured cancer cells derived from HNSCC tissue. Bar: 100 μm. Western blotting ( B ) for p63, ΔNp63, GATA-3, JAM-A, β-catenin, occludin and claudin-1, 4, 7 and flow cytometry ( C ) for JAM-A HNSCC in primary cultured cancer cells transfected with siRNAs of p63, ΔNp63, GATA-3 and JAM-A.

Techniques Used: Staining, Cell Culture, Derivative Assay, Western Blot, Flow Cytometry, Cytometry, Transfection

Western blotting (A) and immunocytochemical staining (B) for JAM-A and β-catenin and flow cytometry (C) for JAM-A in Detroit562 cells treated with various inhibitors GF: PKC inhibitor; LY: PI3K inhibitor; U0126: MAPK inhibitor; iGSK-3β: Wnt inhibitor; AG1478: EGFR inhibitor; SB: p38 MAPK inhibitor; SP: JNK inhibitor; cyclopamine: Hedgehog inhibitor. Bars: 100 μm.
Figure Legend Snippet: Western blotting (A) and immunocytochemical staining (B) for JAM-A and β-catenin and flow cytometry (C) for JAM-A in Detroit562 cells treated with various inhibitors GF: PKC inhibitor; LY: PI3K inhibitor; U0126: MAPK inhibitor; iGSK-3β: Wnt inhibitor; AG1478: EGFR inhibitor; SB: p38 MAPK inhibitor; SP: JNK inhibitor; cyclopamine: Hedgehog inhibitor. Bars: 100 μm.

Techniques Used: Western Blot, Staining, Flow Cytometry, Cytometry

Western blotting (A) and immunocytochemical staining (B) for JAM-A and β-catenin and flow cytometry (C) for JAM-A in Detroit562 cells treated with the NF-κB inhibitors IMD-0354, MG-132 and curcumin. Bars: 100 μm
Figure Legend Snippet: Western blotting (A) and immunocytochemical staining (B) for JAM-A and β-catenin and flow cytometry (C) for JAM-A in Detroit562 cells treated with the NF-κB inhibitors IMD-0354, MG-132 and curcumin. Bars: 100 μm

Techniques Used: Western Blot, Staining, Flow Cytometry, Cytometry, Radial Immuno Diffusion

Western blotting (A, C, E) and immunocytochemical staining (B, D, F) for p63, ΔNp63, JAM-A and β-catenin in Detroit562 cells transfected with siRNAs of p63, ΔNp63 and GATA-3 ( G ) Flow cytometry for JAM-A in Detroit562 cells transfected with siRNAs of p63, ΔNp63 and GATA-3. Bars: 20 μm.
Figure Legend Snippet: Western blotting (A, C, E) and immunocytochemical staining (B, D, F) for p63, ΔNp63, JAM-A and β-catenin in Detroit562 cells transfected with siRNAs of p63, ΔNp63 and GATA-3 ( G ) Flow cytometry for JAM-A in Detroit562 cells transfected with siRNAs of p63, ΔNp63 and GATA-3. Bars: 20 μm.

Techniques Used: Western Blot, Staining, Transfection, Flow Cytometry, Cytometry

Images of H.E. and immunohistochemical staining of MIB1, JAM-A and β-catenin in tissues of HNSCC patients and dysplastic regions ( A ) HNSCC and dysplasia, ( B ) cancer pearl region, ( C ) invasive region, ( D ) metastatic lymph node. Bar: 100 μm. ( E ) Real-time PCR for mRNAs of JAM-A and β-catenin in tonsil and HNSCC-patient tissues. Results are given as means ± SE. ( F ) ELISA for soluble JAM-A in sera of HNSCC patients and healthy control subjects. Results are given as means ±SE. ( F ) p *
Figure Legend Snippet: Images of H.E. and immunohistochemical staining of MIB1, JAM-A and β-catenin in tissues of HNSCC patients and dysplastic regions ( A ) HNSCC and dysplasia, ( B ) cancer pearl region, ( C ) invasive region, ( D ) metastatic lymph node. Bar: 100 μm. ( E ) Real-time PCR for mRNAs of JAM-A and β-catenin in tonsil and HNSCC-patient tissues. Results are given as means ± SE. ( F ) ELISA for soluble JAM-A in sera of HNSCC patients and healthy control subjects. Results are given as means ±SE. ( F ) p *

Techniques Used: Immunohistochemistry, Staining, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

20) Product Images from "Morphological characterization of very small embryonic-like stem cells (VSELs) by ImageStream system analysis"

Article Title: Morphological characterization of very small embryonic-like stem cells (VSELs) by ImageStream system analysis

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/j.1582-4934.2007.00154.x

Gating strategy for sorting VSELs by FACS. Bone marrow (BM)-derived VSELs were isolated from immunofluo-rescence stained full BMMNC population by FACS. Agranular, small events ranging from 2–10 μm were included into gate R1 after comparison with six differently sized beads particles with standard diameters of 1, 2, 4, 6, 10 and 15 μm (Flow Cytometry Size beads, Invitrogen;Molecular Probes, Carlsbad, Ca, USA) ( A ). Bone marrow mononuclear cells (BMMNC) were visualized by dot plots showing FSC (forward scatter) versus SSC (side scatter) signals, which are related to the size and granularity/complexity of the cell, respectively ( B ). Cells from region R1 were further analysed for Sca-1 and Lin expression and only Sca-1 + /Lin − events were included into region R2 ( D ). Population from region R2 was subsequently sorted based on CD45 marker expression into CD45 − and CD45 + subpopulations visualized on histogram ( C ;regions R3 and R4, respectively). Sca-1 + /Lin − /CD45 − cells (VSELs) were sorted as events enclosed in logical gate including regions R1, R2 and R3, while Sca-1 + /Lin − /CD45 + cells (HSCs) from gate including regions R1, R2 and R4. Percentages show the average content of each cellular subpopulation (± S.E.M.) in total BMMNC. Sorted VSELs were re-analysed to establish sorting purity according Lin, CD45 and Sca-1 markers as well as their viability by staining with 7-AAD ( E, F ). Percentages on these two panels present purity of VSELs in each marker and content of viable cells (Mean ± S.E.M.). Cells were not fixed before staining with 7-AAD.
Figure Legend Snippet: Gating strategy for sorting VSELs by FACS. Bone marrow (BM)-derived VSELs were isolated from immunofluo-rescence stained full BMMNC population by FACS. Agranular, small events ranging from 2–10 μm were included into gate R1 after comparison with six differently sized beads particles with standard diameters of 1, 2, 4, 6, 10 and 15 μm (Flow Cytometry Size beads, Invitrogen;Molecular Probes, Carlsbad, Ca, USA) ( A ). Bone marrow mononuclear cells (BMMNC) were visualized by dot plots showing FSC (forward scatter) versus SSC (side scatter) signals, which are related to the size and granularity/complexity of the cell, respectively ( B ). Cells from region R1 were further analysed for Sca-1 and Lin expression and only Sca-1 + /Lin − events were included into region R2 ( D ). Population from region R2 was subsequently sorted based on CD45 marker expression into CD45 − and CD45 + subpopulations visualized on histogram ( C ;regions R3 and R4, respectively). Sca-1 + /Lin − /CD45 − cells (VSELs) were sorted as events enclosed in logical gate including regions R1, R2 and R3, while Sca-1 + /Lin − /CD45 + cells (HSCs) from gate including regions R1, R2 and R4. Percentages show the average content of each cellular subpopulation (± S.E.M.) in total BMMNC. Sorted VSELs were re-analysed to establish sorting purity according Lin, CD45 and Sca-1 markers as well as their viability by staining with 7-AAD ( E, F ). Percentages on these two panels present purity of VSELs in each marker and content of viable cells (Mean ± S.E.M.). Cells were not fixed before staining with 7-AAD.

Techniques Used: FACS, Derivative Assay, Isolation, Staining, Flow Cytometry, Cytometry, Expressing, Marker

N/C ratio and cytoplas-mic area of VSELs. Mean N/C ratios ( A ) and cytoplasmic areas ( B ) of murine BM-derived Sca-1 + /Lin − /CD45 − cells (VSELs) as well as Sca- 1 + /Lin − /CD45 + cells (HSCs), PB-derived granulocytes (PMNCs) and human Nalm-6 cells were measured using the IS. All values are presented as mean (± S.E.M.as calculated by IDEAS software. P
Figure Legend Snippet: N/C ratio and cytoplas-mic area of VSELs. Mean N/C ratios ( A ) and cytoplasmic areas ( B ) of murine BM-derived Sca-1 + /Lin − /CD45 − cells (VSELs) as well as Sca- 1 + /Lin − /CD45 + cells (HSCs), PB-derived granulocytes (PMNCs) and human Nalm-6 cells were measured using the IS. All values are presented as mean (± S.E.M.as calculated by IDEAS software. P

Techniques Used: Derivative Assay, Software

Confocal microscopic images of VSELs and HSCs. Isolated VSELs and HSCs were stained for CD45 (FITC, green fluorescence) and Oct-4 (TRITC, red fluorescence). Nuclei were stained with DAPI (blue fluorescence). ( A ) shows Sca-1 + /Lin − /CD45 + cells (HSCs) that are positive for CD45 and negative for Oct-4. ( B ) shows Sca-1 + /Lin − /CD45 − cell (VSEL), negative for CD45 and positive for Oct-4, a marker of pluripotent cells.
Figure Legend Snippet: Confocal microscopic images of VSELs and HSCs. Isolated VSELs and HSCs were stained for CD45 (FITC, green fluorescence) and Oct-4 (TRITC, red fluorescence). Nuclei were stained with DAPI (blue fluorescence). ( A ) shows Sca-1 + /Lin − /CD45 + cells (HSCs) that are positive for CD45 and negative for Oct-4. ( B ) shows Sca-1 + /Lin − /CD45 − cell (VSEL), negative for CD45 and positive for Oct-4, a marker of pluripotent cells.

Techniques Used: Isolation, Staining, Fluorescence, Marker

Analysis of VSELs and HSCs according to the size. Sca-1 + /Lin − /CD45 − cells (VSELs) and Sca-1 + /Lin − /CD45 + cells (HSCs) were identified and analysed by FACS as shown previously. B and C shows the size analysis of Sca-1 + /Lin − /CD45 − cells (VSELs; blue) and Sca-1 + /Lin − /CD45 + cells (HSCs; red) in comparison to ( A ). In accordance with bead size, the blue, black and red boxes on dot-plots contain events between 2 and 6 μm (region R3), 6–10 μm events (region R4), and > 6 μm (region R5), respectively. ( B ) The size analysis of only small cells enclosed in region R1 (see Fig. 1B ); right graph presents the percent of VSELs (blue bar) and HSCs (red bar) with the cell size between 2 and 6 μm. The majority of VSELs is enclosed in region R3 (blue box), while HSCs belong to region R4 (black box) containing events between 6 and 10 μm (left panel).( C ) shows the size analysis of total BMMNC. Graph on the right presents the percentage of cells with VSELs'phenotype (blue bar) and HSCs' phenotype (red bar) with the size between 2 and 6 μm. The majority of HSCs belong to region R5 (red box) containing events larger than 6μm(left panel). All values are presented as mean (SEM). P -values less than 0.05 are considered statistically significant (*).
Figure Legend Snippet: Analysis of VSELs and HSCs according to the size. Sca-1 + /Lin − /CD45 − cells (VSELs) and Sca-1 + /Lin − /CD45 + cells (HSCs) were identified and analysed by FACS as shown previously. B and C shows the size analysis of Sca-1 + /Lin − /CD45 − cells (VSELs; blue) and Sca-1 + /Lin − /CD45 + cells (HSCs; red) in comparison to ( A ). In accordance with bead size, the blue, black and red boxes on dot-plots contain events between 2 and 6 μm (region R3), 6–10 μm events (region R4), and > 6 μm (region R5), respectively. ( B ) The size analysis of only small cells enclosed in region R1 (see Fig. 1B ); right graph presents the percent of VSELs (blue bar) and HSCs (red bar) with the cell size between 2 and 6 μm. The majority of VSELs is enclosed in region R3 (blue box), while HSCs belong to region R4 (black box) containing events between 6 and 10 μm (left panel).( C ) shows the size analysis of total BMMNC. Graph on the right presents the percentage of cells with VSELs'phenotype (blue bar) and HSCs' phenotype (red bar) with the size between 2 and 6 μm. The majority of HSCs belong to region R5 (red box) containing events larger than 6μm(left panel). All values are presented as mean (SEM). P -values less than 0.05 are considered statistically significant (*).

Techniques Used: FACS

Identification of size of VSELs and HSCs by ImageStream system. Cellular diameter analysis ( A ) and representative images ( B ) of murine and human cells illustrate for their size and morphology. Panel A presents comparison of cellular diameter between murine BM-derived Sca-1 + /Lin − /CD45 − (VSELs), Sca-1 + /Lin − /CD45 + (HSCs), polymor-phonuclear cells (PMNCs) isolated from blood and human leukaemia B cell (Nalm-6 cell line). Size of the cells was calculated based on the scale measurements employed by IDEAS software. Graph and table include mean ± S.E.M. P
Figure Legend Snippet: Identification of size of VSELs and HSCs by ImageStream system. Cellular diameter analysis ( A ) and representative images ( B ) of murine and human cells illustrate for their size and morphology. Panel A presents comparison of cellular diameter between murine BM-derived Sca-1 + /Lin − /CD45 − (VSELs), Sca-1 + /Lin − /CD45 + (HSCs), polymor-phonuclear cells (PMNCs) isolated from blood and human leukaemia B cell (Nalm-6 cell line). Size of the cells was calculated based on the scale measurements employed by IDEAS software. Graph and table include mean ± S.E.M. P

Techniques Used: Derivative Assay, Isolation, Software

Analysis of nuclear to cytoplasmic ratio by ImageStream system. Single, round cells from region R1 ( A ) were visualized based on their nuclear to cytoplasm ratio and Lin markers expression (X- and Y- axis, respectively ( B ). Cellular populations were gated including Lin + cells with low nuclear to cytoplasmic ratio (0.936 ± 0.016) (region R3, red) and Lin − cells with high N/C ratio (3.485 ± 0.248) (region R2, orange). Objects from region R2 were farther analysed for their CD45 and Sca-1 expression (X- and Y-axis, respectively ( B ). Cells with VSELs' phenotype (Sca-1 + /Lin − /CD45 − ) and characterized by higher N/C ratio (1.471(0.171) were included in region R4 (magenta; C ) and visualized on the other plots as diamonds (magenta). N/C ratio was calculated as nuclear area divided by cytoplasmic area computed from nuclear (7-AAD) and brightfield images. Signals of brightfield, Lin-PE and 7-AAD were collected by the IS in channels 2, 4 and 5, respectively. Mean (± S.E.M.) values of N/C ratio were calculated using IDEAS software.
Figure Legend Snippet: Analysis of nuclear to cytoplasmic ratio by ImageStream system. Single, round cells from region R1 ( A ) were visualized based on their nuclear to cytoplasm ratio and Lin markers expression (X- and Y- axis, respectively ( B ). Cellular populations were gated including Lin + cells with low nuclear to cytoplasmic ratio (0.936 ± 0.016) (region R3, red) and Lin − cells with high N/C ratio (3.485 ± 0.248) (region R2, orange). Objects from region R2 were farther analysed for their CD45 and Sca-1 expression (X- and Y-axis, respectively ( B ). Cells with VSELs' phenotype (Sca-1 + /Lin − /CD45 − ) and characterized by higher N/C ratio (1.471(0.171) were included in region R4 (magenta; C ) and visualized on the other plots as diamonds (magenta). N/C ratio was calculated as nuclear area divided by cytoplasmic area computed from nuclear (7-AAD) and brightfield images. Signals of brightfield, Lin-PE and 7-AAD were collected by the IS in channels 2, 4 and 5, respectively. Mean (± S.E.M.) values of N/C ratio were calculated using IDEAS software.

Techniques Used: Expressing, Software

Identification of VSELs by ImageStream system. The ImageStream system software (IDEAS) identified Sca-1 + /Lin − /CD45 − ( A ) and visualized an image gallery of the objects identified by this phenotype ( B ). BMMNC were stained for CD45, Sca-1 and Lin markers, fixed with paraformaldehyde solution and analysed. Signals from CD45-FITC, Lin-PE, 7-AAD and Sca-1-PE-Cy5 were collected by channels 3, 4, 5 and 6, respectively. Side scatter and brightfield were detected by channels 1 and 2, respectively. The dot-plot ( A ) shows all objects according to area of brightfield (Ch2), related to size of objects (X-axis) and aspect ratio of brightfield related to shape of objects (Y-axis). The aspect ratio was calculated based on brightfield as the ratio of cellular minor axis (width) to major axis (height). Round, non-elongated cells have aspect ratio close to 1.0, while the elongated cells or clumps had lower aspect ratio. When applied to bone marrow cells, region R1 encloses mostly single, round objects resembling cells. Subsequently, objects from region R1 are visualized according to their CD45 and Lin expression (X- and Y- axis, respectively; A , middle dot-plot). CD45 − /Lin − objects were included into region R2 and further analysed based on Sca-1 expression ( A , lower histogram). ( B ) shows the image gallery of nucleated objects included into region R3 and defined as Sca-1 + /Lin − /CD45 − . Cells were fixed before staining with 7-AAD.
Figure Legend Snippet: Identification of VSELs by ImageStream system. The ImageStream system software (IDEAS) identified Sca-1 + /Lin − /CD45 − ( A ) and visualized an image gallery of the objects identified by this phenotype ( B ). BMMNC were stained for CD45, Sca-1 and Lin markers, fixed with paraformaldehyde solution and analysed. Signals from CD45-FITC, Lin-PE, 7-AAD and Sca-1-PE-Cy5 were collected by channels 3, 4, 5 and 6, respectively. Side scatter and brightfield were detected by channels 1 and 2, respectively. The dot-plot ( A ) shows all objects according to area of brightfield (Ch2), related to size of objects (X-axis) and aspect ratio of brightfield related to shape of objects (Y-axis). The aspect ratio was calculated based on brightfield as the ratio of cellular minor axis (width) to major axis (height). Round, non-elongated cells have aspect ratio close to 1.0, while the elongated cells or clumps had lower aspect ratio. When applied to bone marrow cells, region R1 encloses mostly single, round objects resembling cells. Subsequently, objects from region R1 are visualized according to their CD45 and Lin expression (X- and Y- axis, respectively; A , middle dot-plot). CD45 − /Lin − objects were included into region R2 and further analysed based on Sca-1 expression ( A , lower histogram). ( B ) shows the image gallery of nucleated objects included into region R3 and defined as Sca-1 + /Lin − /CD45 − . Cells were fixed before staining with 7-AAD.

Techniques Used: Software, Staining, Expressing

21) Product Images from "CD8+ T Cells and IFN-? Mediate the Time-Dependent Accumulation of Infected Red Blood Cells in Deep Organs during Experimental Cerebral Malaria"

Article Title: CD8+ T Cells and IFN-? Mediate the Time-Dependent Accumulation of Infected Red Blood Cells in Deep Organs during Experimental Cerebral Malaria

Journal: PLoS ONE

doi: 10.1371/journal.pone.0018720

Depletion of myeloid cells does not alter IRBC distribution during ECM. MAFIA mice were infected with PbA luc and injected on days 5, 6, and 7 with the drug AP20187 as described in Material and Methods . (A) Depletion of granulocytes/monocytes (defined as CD45 + CD11b + Gr1 + ) was assessed by flow cytometry on day 7 post-infection. Data plots presented are from one mouse and similar data were obtained for 4 more mice. (B) Survival and (C) parasitemia of treated and non-treated MAFIA mice (5 per group). Neurologic signs of CM appeared on days 6–12 (shaded area), with death occurring 24–48 h after onset. Parasitemia (%) values are expressed as mean ± SD of 5 mice per group. In vivo bioluminescence imaging quantification of IRBC accumulation in the whole body (D) and head (E) of treated and non-treated infected mice. Luminescence values (log) as mean ± SD of 5 mice.
Figure Legend Snippet: Depletion of myeloid cells does not alter IRBC distribution during ECM. MAFIA mice were infected with PbA luc and injected on days 5, 6, and 7 with the drug AP20187 as described in Material and Methods . (A) Depletion of granulocytes/monocytes (defined as CD45 + CD11b + Gr1 + ) was assessed by flow cytometry on day 7 post-infection. Data plots presented are from one mouse and similar data were obtained for 4 more mice. (B) Survival and (C) parasitemia of treated and non-treated MAFIA mice (5 per group). Neurologic signs of CM appeared on days 6–12 (shaded area), with death occurring 24–48 h after onset. Parasitemia (%) values are expressed as mean ± SD of 5 mice per group. In vivo bioluminescence imaging quantification of IRBC accumulation in the whole body (D) and head (E) of treated and non-treated infected mice. Luminescence values (log) as mean ± SD of 5 mice.

Techniques Used: Mouse Assay, Infection, Injection, Flow Cytometry, Cytometry, In Vivo, Imaging

22) Product Images from "Endogenous IL-12 triggers an antiangiogenic program in melanoma cells"

Article Title: Endogenous IL-12 triggers an antiangiogenic program in melanoma cells

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.0609028104

Characterization and tumorigenicity of B16 cells and IL12p70 serum level in KO mice. ( A ) Expression of IL12rb1 and b2 chains in B16 melanoma cells. ( Left ) IL12rb1 surface expression in B16 cells, as assessed by flow cytometry. Open profile: IL12rb1 staining; dark profile: isotype matched mAb staining. ( Right ) IL12rb2 expression in B16 cells, as assessed by RT-PCR. MW, molecular weight; NC, negative control (water in the place of cDNA); PC, positive control (3T3 cell line); B16 cells, B16 melanoma cells (B16). On the right, the expected mw of the amplified band is shown. ( B ) Quantitative determination of IL-12p70 concentrations in sera from 10 KO mice, as assessed by ELISA. ( C ) Volume of s.c. tumors grown in KO and WT animals 17 days ( Left ) and 7 days ( Right ) after B16 cell inoculation. The differences in size between tumors removed from KO and WT mice either 17 days or 7 days after B16 cell inoculation were evaluated by Mann–Whitney U test. Boxes indicate values between the 25 th and 75 th percentiles, whisker lines represent highest and lowest values for each group. Horizontal lines represent median values.
Figure Legend Snippet: Characterization and tumorigenicity of B16 cells and IL12p70 serum level in KO mice. ( A ) Expression of IL12rb1 and b2 chains in B16 melanoma cells. ( Left ) IL12rb1 surface expression in B16 cells, as assessed by flow cytometry. Open profile: IL12rb1 staining; dark profile: isotype matched mAb staining. ( Right ) IL12rb2 expression in B16 cells, as assessed by RT-PCR. MW, molecular weight; NC, negative control (water in the place of cDNA); PC, positive control (3T3 cell line); B16 cells, B16 melanoma cells (B16). On the right, the expected mw of the amplified band is shown. ( B ) Quantitative determination of IL-12p70 concentrations in sera from 10 KO mice, as assessed by ELISA. ( C ) Volume of s.c. tumors grown in KO and WT animals 17 days ( Left ) and 7 days ( Right ) after B16 cell inoculation. The differences in size between tumors removed from KO and WT mice either 17 days or 7 days after B16 cell inoculation were evaluated by Mann–Whitney U test. Boxes indicate values between the 25 th and 75 th percentiles, whisker lines represent highest and lowest values for each group. Horizontal lines represent median values.

Techniques Used: Mouse Assay, Expressing, Flow Cytometry, Cytometry, Staining, Reverse Transcription Polymerase Chain Reaction, Molecular Weight, Negative Control, Positive Control, Amplification, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Whisker Assay

23) Product Images from "The Expression of Formyl Peptide Receptor 1 is Correlated with Tumor Invasion of Human Colorectal Cancer"

Article Title: The Expression of Formyl Peptide Receptor 1 is Correlated with Tumor Invasion of Human Colorectal Cancer

Journal: Scientific Reports

doi: 10.1038/s41598-017-06368-9

FPR1 activation promoted SW480 cell migration. SW480 ( a ) and HT29 ( b ) were treated with fMLF (1 μM) or 20% FBS (positive control) for 12 h with or without a 15 min pretreatment with cyclosporine H (1 μM). The cell migration was examined using a 48-well Boyden chamber. Representative images of migrated cells (SW480 and TH29) on membrane filters were shown in ( a and b ) and quantified data were shown in ( c and d ), respectively. * p
Figure Legend Snippet: FPR1 activation promoted SW480 cell migration. SW480 ( a ) and HT29 ( b ) were treated with fMLF (1 μM) or 20% FBS (positive control) for 12 h with or without a 15 min pretreatment with cyclosporine H (1 μM). The cell migration was examined using a 48-well Boyden chamber. Representative images of migrated cells (SW480 and TH29) on membrane filters were shown in ( a and b ) and quantified data were shown in ( c and d ), respectively. * p

Techniques Used: Activation Assay, Migration, Positive Control

Immunofluorescence staining of FPR1 in human CRC tissues. Frozen sections of the adjacent normal tissues ( a ) and CRC tissues ( b ) were stained by immunofluorescence for the expression of FPR1 (FITC, green) and MPO (Cy3, red), as described in Materials and Methods . Cell nuclei were stained with DAPI (blue). Images shown are representative of three independent experiments with similar results. Scale bar, 25 µm.
Figure Legend Snippet: Immunofluorescence staining of FPR1 in human CRC tissues. Frozen sections of the adjacent normal tissues ( a ) and CRC tissues ( b ) were stained by immunofluorescence for the expression of FPR1 (FITC, green) and MPO (Cy3, red), as described in Materials and Methods . Cell nuclei were stained with DAPI (blue). Images shown are representative of three independent experiments with similar results. Scale bar, 25 µm.

Techniques Used: Immunofluorescence, Staining, Expressing

Expression of FPR1 in colorectal cancer cell lines. The mRNA transcripts ( a ) and protein expression ( b ) of FPR1 in SW480 and HT29 were analyzed by agarose gel electrophoresis and western blotting, respectively. The full-length gels and blots were presented in Supplementary Fig. S4a and b . ( c – f ) Flow cytometry was used to confirm the distribution of FPR1 expression in these two cell lines. The cells were treated with or without membrane-penetration solution to detect the total protein expression ( d , f ) and membrane expression ( c , e ).
Figure Legend Snippet: Expression of FPR1 in colorectal cancer cell lines. The mRNA transcripts ( a ) and protein expression ( b ) of FPR1 in SW480 and HT29 were analyzed by agarose gel electrophoresis and western blotting, respectively. The full-length gels and blots were presented in Supplementary Fig. S4a and b . ( c – f ) Flow cytometry was used to confirm the distribution of FPR1 expression in these two cell lines. The cells were treated with or without membrane-penetration solution to detect the total protein expression ( d , f ) and membrane expression ( c , e ).

Techniques Used: Expressing, Agarose Gel Electrophoresis, Western Blot, Flow Cytometry, Cytometry

FPR1 activation accelerated the migration of SW480. The motility of SW480 ( a , b ) and HT29 ( c , d ) in wound-healing model was assayed in the presence of fMLF (1 μM) or 10% FBS with or without a 15 min pretreatment with cyclosporine H (1 μM). Then cells were photographed at 24 h and 48 h. * p
Figure Legend Snippet: FPR1 activation accelerated the migration of SW480. The motility of SW480 ( a , b ) and HT29 ( c , d ) in wound-healing model was assayed in the presence of fMLF (1 μM) or 10% FBS with or without a 15 min pretreatment with cyclosporine H (1 μM). Then cells were photographed at 24 h and 48 h. * p

Techniques Used: Activation Assay, Migration

Expression of FPRs was increased in CRC tissues. ( a ) The levels of FPR mRNA in CRC tissues, distant control, and adjacent non-tumor tissues were examined with real-time quantitative PCR. The relative transcript expression was calculated as 2 −ΔΔCT and was normalized against GAPDH/PPIA. ( b ) The tissue homogenates were analyzed by western blotting for the expression of FPR1. Two representative sample blots (p5 and p13) are shown. M, distant mormal tissue; N, adjacent non-tumor tissue; T, cancer tissue. The full-length blots were presented in Supplementary Fig. S3 . ( c ) The blots were quantified densitometrically and the relative immunoreactivities are shown. All data shown are means ± SEM. * p
Figure Legend Snippet: Expression of FPRs was increased in CRC tissues. ( a ) The levels of FPR mRNA in CRC tissues, distant control, and adjacent non-tumor tissues were examined with real-time quantitative PCR. The relative transcript expression was calculated as 2 −ΔΔCT and was normalized against GAPDH/PPIA. ( b ) The tissue homogenates were analyzed by western blotting for the expression of FPR1. Two representative sample blots (p5 and p13) are shown. M, distant mormal tissue; N, adjacent non-tumor tissue; T, cancer tissue. The full-length blots were presented in Supplementary Fig. S3 . ( c ) The blots were quantified densitometrically and the relative immunoreactivities are shown. All data shown are means ± SEM. * p

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Western Blot

FPR1 involvement in mouse survival in a colorectal cancer model. ( a ) Schematic representation of the mouse model of CRC, as described in the Materials and Methods section. Body weights ( b ) and survival ( c ) of WT (green line) mice and fpr1 −/− (red line) mice with CRC. Also shown are quantification of colon length ( d ) and tumor number ( e ) in WT mice and fpr1 −/− mice with CRC. (6 mice for WT group; 13 mice for fpr1 −/− group).
Figure Legend Snippet: FPR1 involvement in mouse survival in a colorectal cancer model. ( a ) Schematic representation of the mouse model of CRC, as described in the Materials and Methods section. Body weights ( b ) and survival ( c ) of WT (green line) mice and fpr1 −/− (red line) mice with CRC. Also shown are quantification of colon length ( d ) and tumor number ( e ) in WT mice and fpr1 −/− mice with CRC. (6 mice for WT group; 13 mice for fpr1 −/− group).

Techniques Used: Mouse Assay

24) Product Images from "Anti-JNK2 peptide–siRNA nanostructures improve plaque endothelium and reduce thrombotic risk in atherosclerotic mice"

Article Title: Anti-JNK2 peptide–siRNA nanostructures improve plaque endothelium and reduce thrombotic risk in atherosclerotic mice

Journal: International Journal of Nanomedicine

doi: 10.2147/IJN.S168556

p5RHH-JNK2 siRNA NPs neither suppress systemic immune-cell function nor induce innate/adaptive immunoresponses. Notes: ( A ) After seven sequential doses of p5RHH-siRNA NPs over 3.5 weeks, spleens were extracted and splenocytes enumerated 24 hours after the last dose. ApoE −/- mice treated with p5RHH-JNK2 NPs (n=4) exhibit significantly fewer splenocytes compared to HBSS control (n=3) ( P =0.02). ( B ) Spleen sizes of mice treated with p5RHH-JNK2 siRNA NPs (n=19) are significantly smaller than from mice with HBSS treatment (n=12, P =0.001) and approximate spleen sizes of control C57BL/6 mice (n=9, P =0.511; one-way ANOVA followed with Scheffé post hoc test). ( C ) Distribution of splenic immune-cell subpopulations was not affected by the p5RHH-JNK2 siRNA NP treatment (n=4) compared to HBSS control (n=3; FoxP3 + p5RHH-JNK2 siRNA NP treatment, n=6; HBBS, n=5). ( D ) Splenic CD4 + T cells stimulated with anti-CD3 monoclonal antibody responded normally (HBSS, n=5; p5RHH-JNK2 siRNA NPs, n=6). ( E ) C3a assay indicates that p5RHH-JNK2 siRNA NPs (n=5) do not activate complement (innate immune response) compared with DOTAP NPs known to activate strongly (n=5). C3a level of the mice treated with the p5RHH-JNK2 siRNA NPs is significantly smaller than those of mice treated with DOTAP NP ( P
Figure Legend Snippet: p5RHH-JNK2 siRNA NPs neither suppress systemic immune-cell function nor induce innate/adaptive immunoresponses. Notes: ( A ) After seven sequential doses of p5RHH-siRNA NPs over 3.5 weeks, spleens were extracted and splenocytes enumerated 24 hours after the last dose. ApoE −/- mice treated with p5RHH-JNK2 NPs (n=4) exhibit significantly fewer splenocytes compared to HBSS control (n=3) ( P =0.02). ( B ) Spleen sizes of mice treated with p5RHH-JNK2 siRNA NPs (n=19) are significantly smaller than from mice with HBSS treatment (n=12, P =0.001) and approximate spleen sizes of control C57BL/6 mice (n=9, P =0.511; one-way ANOVA followed with Scheffé post hoc test). ( C ) Distribution of splenic immune-cell subpopulations was not affected by the p5RHH-JNK2 siRNA NP treatment (n=4) compared to HBSS control (n=3; FoxP3 + p5RHH-JNK2 siRNA NP treatment, n=6; HBBS, n=5). ( D ) Splenic CD4 + T cells stimulated with anti-CD3 monoclonal antibody responded normally (HBSS, n=5; p5RHH-JNK2 siRNA NPs, n=6). ( E ) C3a assay indicates that p5RHH-JNK2 siRNA NPs (n=5) do not activate complement (innate immune response) compared with DOTAP NPs known to activate strongly (n=5). C3a level of the mice treated with the p5RHH-JNK2 siRNA NPs is significantly smaller than those of mice treated with DOTAP NP ( P

Techniques Used: Cell Function Assay, Mouse Assay

25) Product Images from "Resistance to Streptozotocin-Induced Autoimmune Diabetes in Absence of Complement C3: Myeloid-Derived Suppressor Cells Play a Role"

Article Title: Resistance to Streptozotocin-Induced Autoimmune Diabetes in Absence of Complement C3: Myeloid-Derived Suppressor Cells Play a Role

Journal: PLoS ONE

doi: 10.1371/journal.pone.0066334

MDSC induction in C3−/− mice subjected to STZ is TGF-β-dependent. ( A ) the plasma from C3+/+ (n = 8) or C3−/− mice (n = 15) was collected 3 weeks after STZ treatment. TGF-β contents were examined by ELISA. ( B,C ) TGF-β antibody or isotype IgG was adminidtrated to STZ-treated C3−/− mice as described in Materials and methods. ( B ) blood glucose was monitored and the percentage of diabetes-free survival was expressed. Data were pooled from three independent experiments. Each group consists of 10–15 mice. ( C ) 4 weeks later, spleen cells were fractionated. The percentage and number of Gr-1 + CD11b + cells was detected by flow cytometry. Representative data were shown from three separate experiments. ( D,E ) anti-CD25 antibody or isotype IgG was injected into STZ-treated C3−/− mice as described in Materials and methods. ( D ) diabetes-free survival was monitored every week. Each experiment group consisted of 10–15 mice. ( E ) 4 weeks later, the percentage and number of Gr-1 + CD11b + cells was examined by flow cytometry. Representative data from two separate experiments were shown. Number is 5 to 8 per group when the abundance of MDSC was calculated. **, P
Figure Legend Snippet: MDSC induction in C3−/− mice subjected to STZ is TGF-β-dependent. ( A ) the plasma from C3+/+ (n = 8) or C3−/− mice (n = 15) was collected 3 weeks after STZ treatment. TGF-β contents were examined by ELISA. ( B,C ) TGF-β antibody or isotype IgG was adminidtrated to STZ-treated C3−/− mice as described in Materials and methods. ( B ) blood glucose was monitored and the percentage of diabetes-free survival was expressed. Data were pooled from three independent experiments. Each group consists of 10–15 mice. ( C ) 4 weeks later, spleen cells were fractionated. The percentage and number of Gr-1 + CD11b + cells was detected by flow cytometry. Representative data were shown from three separate experiments. ( D,E ) anti-CD25 antibody or isotype IgG was injected into STZ-treated C3−/− mice as described in Materials and methods. ( D ) diabetes-free survival was monitored every week. Each experiment group consisted of 10–15 mice. ( E ) 4 weeks later, the percentage and number of Gr-1 + CD11b + cells was examined by flow cytometry. Representative data from two separate experiments were shown. Number is 5 to 8 per group when the abundance of MDSC was calculated. **, P

Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cytometry, Injection

26) Product Images from "Blood Flukes Exploit Peyer's Patch Lymphoid Tissue to Facilitate Transmission from the Mammalian Host"

Article Title: Blood Flukes Exploit Peyer's Patch Lymphoid Tissue to Facilitate Transmission from the Mammalian Host

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1003063

Egg deposition within PP disrupts lymphoid microarchitecture and causes loss of cellularity. (A) DsRed and eYFP expression demarcate B cell follicles and inter-follicular T cell zones of PP. Images are 3D rendered multi-photon confocal display of excised PP tissue derived from VaDsRed/CD19+ eYFP double fluorescent reporter mice (red = CD3+ cells; green = CD19+ cells; blue = second-harmonic generation by collagen fibres). Auto-fluorescent egg (arrows) proximal to PP at 6 and 8 weeks pi. All data are representative of two independent experiments, n = 3 mice. (B) Stereo fluorescent stereomicroscope images of PP in naïve, and infected CD3+/CD19+ reporter mice. Auto-fluorescent eggs (arrows) co-localise to T and B cell zones of PP at 14 weeks (lower panel). (C) 3D multiphoton confocal image of PP egg-granuloma (enlargement of section inset shown in B as dotted line); Sm = autofluorescent egg; arrows = cellular infiltrate; blue = second-harmonic generation by collagen fibres. (D) Numbers of CD11b+ cells, CD11b+SigLecF-F4/80+ macrophages, and CD11b+SigLecF+F4/80lo eosinophils in PP cell suspensions enumerated by flow cytometry in naïve mice, or after infection. (E) CD19+ PP follicle area in double reporter mice (min 3 PP/mouse). (F) Total viable PP, B220+ and IgA+B220+ B cell yields/mouse and (G) proportion of B220+ cells of total PP. (H) Stereo fluorescent stereomicroscope images of mLN in situ from naïve and infected CD3+ (red)/CD19+ (green) reporter mice. (I) mLN follicle area, (J) total viable cell and B220+ B cell yields in the mLN of naïve and infected CD3+T cell/CD19+B cell double reporter mice. All data; *P
Figure Legend Snippet: Egg deposition within PP disrupts lymphoid microarchitecture and causes loss of cellularity. (A) DsRed and eYFP expression demarcate B cell follicles and inter-follicular T cell zones of PP. Images are 3D rendered multi-photon confocal display of excised PP tissue derived from VaDsRed/CD19+ eYFP double fluorescent reporter mice (red = CD3+ cells; green = CD19+ cells; blue = second-harmonic generation by collagen fibres). Auto-fluorescent egg (arrows) proximal to PP at 6 and 8 weeks pi. All data are representative of two independent experiments, n = 3 mice. (B) Stereo fluorescent stereomicroscope images of PP in naïve, and infected CD3+/CD19+ reporter mice. Auto-fluorescent eggs (arrows) co-localise to T and B cell zones of PP at 14 weeks (lower panel). (C) 3D multiphoton confocal image of PP egg-granuloma (enlargement of section inset shown in B as dotted line); Sm = autofluorescent egg; arrows = cellular infiltrate; blue = second-harmonic generation by collagen fibres. (D) Numbers of CD11b+ cells, CD11b+SigLecF-F4/80+ macrophages, and CD11b+SigLecF+F4/80lo eosinophils in PP cell suspensions enumerated by flow cytometry in naïve mice, or after infection. (E) CD19+ PP follicle area in double reporter mice (min 3 PP/mouse). (F) Total viable PP, B220+ and IgA+B220+ B cell yields/mouse and (G) proportion of B220+ cells of total PP. (H) Stereo fluorescent stereomicroscope images of mLN in situ from naïve and infected CD3+ (red)/CD19+ (green) reporter mice. (I) mLN follicle area, (J) total viable cell and B220+ B cell yields in the mLN of naïve and infected CD3+T cell/CD19+B cell double reporter mice. All data; *P

Techniques Used: Expressing, Derivative Assay, Mouse Assay, Infection, Flow Cytometry, Cytometry, In Situ

PP are required to sustain optimum egg transmission and limit host morbidity. (A) Quantitative analysis of adult schistosome worm recoveries following perfusion of the hepatic portal system in WT or PP null mice. (B) Cytokine secretion by mLN cells from WT and PP null mice 8 weeks post-infection following in vitro stimulation with anti-CD3 mAb, or soluble egg antigen. (C) Masson's Trichrome stained histological sections of duodenum (blue = collagen) at post-infection in WT or PP null mice (red dotted lines = extent of egg granulomas). (D) Reduction in granuloma areas at 16 cf. 8 weeks; min 4 granulomas/mouse, **P
Figure Legend Snippet: PP are required to sustain optimum egg transmission and limit host morbidity. (A) Quantitative analysis of adult schistosome worm recoveries following perfusion of the hepatic portal system in WT or PP null mice. (B) Cytokine secretion by mLN cells from WT and PP null mice 8 weeks post-infection following in vitro stimulation with anti-CD3 mAb, or soluble egg antigen. (C) Masson's Trichrome stained histological sections of duodenum (blue = collagen) at post-infection in WT or PP null mice (red dotted lines = extent of egg granulomas). (D) Reduction in granuloma areas at 16 cf. 8 weeks; min 4 granulomas/mouse, **P

Techniques Used: Transmission Assay, Mouse Assay, Infection, In Vitro, Staining

27) Product Images from "Fetal thymus graft enables recovery from age-related hearing loss and expansion of CD4-Positive T cells expressing IL-1 receptor type 2 and regulatory T Cells"

Article Title: Fetal thymus graft enables recovery from age-related hearing loss and expansion of CD4-Positive T cells expressing IL-1 receptor type 2 and regulatory T Cells

Journal: Immunity & Ageing : I & A

doi: 10.1186/s12979-015-0053-9

Gating for IL-1R1, IL-1R2, and FR4 on CD4 + T cells. Surface markers of IL-1R1 ( a ), IL-1R2 ( b ), and FR4 ( c ) on CD4 + T cells were examined using FITC-conjugated anti-mouse CD4 cell mAb, PE-conjugated anti-mouse CD121a (IL-1R1) mAb, PE-conjugated anti-mouse CD121b (IL-1R2) mAb, and biotin-conjugated anti-mouse FR4 (nTreg) mAb plus streptavidin-RED670 with flow cytometry. a) Square R1, b) square R2, and c) square R3 include CD121a + CD4 + cells, CD121b + CD4 + cells, and FR4 hi CD4 + cells, respectively
Figure Legend Snippet: Gating for IL-1R1, IL-1R2, and FR4 on CD4 + T cells. Surface markers of IL-1R1 ( a ), IL-1R2 ( b ), and FR4 ( c ) on CD4 + T cells were examined using FITC-conjugated anti-mouse CD4 cell mAb, PE-conjugated anti-mouse CD121a (IL-1R1) mAb, PE-conjugated anti-mouse CD121b (IL-1R2) mAb, and biotin-conjugated anti-mouse FR4 (nTreg) mAb plus streptavidin-RED670 with flow cytometry. a) Square R1, b) square R2, and c) square R3 include CD121a + CD4 + cells, CD121b + CD4 + cells, and FR4 hi CD4 + cells, respectively

Techniques Used: Flow Cytometry, Cytometry

28) Product Images from "M tuberculosis in the Adjuvant Modulates Time of Appearance of CNS-Specific Effector T Cells in the Spleen through a Polymorphic Site of TLR2"

Article Title: M tuberculosis in the Adjuvant Modulates Time of Appearance of CNS-Specific Effector T Cells in the Spleen through a Polymorphic Site of TLR2

Journal: PLoS ONE

doi: 10.1371/journal.pone.0055819

Identification of polymorphisms of TLR2 between SJL and B6 strains and expression of TLR2 on immune cells. A ) Comparison of the sequence of TLR2 of SJL and B6. The non-synonymous and synonymous polymorphisms are boxed. Base (first line) and aminoacid (second line) sequences of B6 and the corresponding sequences (third and fourth lines, respectively) of SJL around the polymorphic residues are reported. B ) Identification of enriched activated T cells, naïve T cells and APC in LN cells by scattering properties. SJL, B6 wt , F1 (SJLxB6 wt ) and F1 (SJLxB6 tlr2− ) mice were immunized with IFA containing 50 microgrammes/mouse of M tb .. 8 days later, mice were sacrificed and cells from draining LN were loaded with CFSE and stimulated with PPD. After 3 days, cells were collected and stained with PE-labeled anti-CD3 monoclonal antibody. The colours define cells examined for TLR2 expression in panel C. C ) SJL, B6 wt , F1 (SJLxB6 wt ) and F1 (SJLxB6 tlr2− ) mice were immunized with IFA containing 50 microgrammes/mouse of M tb . Eight d later, mice were sacrificed and cells from draining LN were stimulated with PPD. After 3 days, cells were collected and stained with PE-labeled anti-CD3 mAb and with FITC labeled anti-TLR2 mAb. Expression of TLR2 was evaluated on high scattering CD3 + cells (activated T cells, red line), low scattering CD3 + cells (naïve T cells, shaded area) and CD3 − cells (mostly APC, black line), as shown in B .
Figure Legend Snippet: Identification of polymorphisms of TLR2 between SJL and B6 strains and expression of TLR2 on immune cells. A ) Comparison of the sequence of TLR2 of SJL and B6. The non-synonymous and synonymous polymorphisms are boxed. Base (first line) and aminoacid (second line) sequences of B6 and the corresponding sequences (third and fourth lines, respectively) of SJL around the polymorphic residues are reported. B ) Identification of enriched activated T cells, naïve T cells and APC in LN cells by scattering properties. SJL, B6 wt , F1 (SJLxB6 wt ) and F1 (SJLxB6 tlr2− ) mice were immunized with IFA containing 50 microgrammes/mouse of M tb .. 8 days later, mice were sacrificed and cells from draining LN were loaded with CFSE and stimulated with PPD. After 3 days, cells were collected and stained with PE-labeled anti-CD3 monoclonal antibody. The colours define cells examined for TLR2 expression in panel C. C ) SJL, B6 wt , F1 (SJLxB6 wt ) and F1 (SJLxB6 tlr2− ) mice were immunized with IFA containing 50 microgrammes/mouse of M tb . Eight d later, mice were sacrificed and cells from draining LN were stimulated with PPD. After 3 days, cells were collected and stained with PE-labeled anti-CD3 mAb and with FITC labeled anti-TLR2 mAb. Expression of TLR2 was evaluated on high scattering CD3 + cells (activated T cells, red line), low scattering CD3 + cells (naïve T cells, shaded area) and CD3 − cells (mostly APC, black line), as shown in B .

Techniques Used: Expressing, Sequencing, Mouse Assay, Immunofluorescence, Staining, Labeling

Mobilization of T cells is strain and TLR2-dependent. F1 (SJLxB6 wt ), F1 (SJLxB6 tlr− ) ( A , C ) or SJL (B, D ) mice were immunized with p139 in IFA in the presence of the indicated amounts of killed M tb ( A ), or of PPD ( B , C ) or of a 1∶1 w/w mixture of PAM2-(CSK)3 and PAM3-(CSK)3 ( D ). The number of mice for each group is indicated in the figure. Fourteen days later mice were sacrificed and the presence of T cells carrying the public TCR-beta chain in LN (closed symbols) and spleen (open symbols) was measured by immunoscope. Data are reported as RSI for the peak corresponding to the public BV10 TCR-beta chain for each individual mouse. Dashed lines indicate the cut off value for positivity (established as described in Results). E : 4 F1 (SJLxB6 wt , circles) mice and 5 F1 (SJLxB6 tlr2− , triangles) mice were immunized with regular CFA in PBS. Two weeks later cells from draining LN (full symbols) and spleen (open symbols) were recovered, labeled with CFSE and cultured in the presence or absence (background) of PPD. After 3 days, the number of CD3 + cells that had specifically divided in response to PPD was determined as % of CFSE low CD3 + cells over total CD3 + cells in the PPD stimulated sample minus % of CFSE low CD3 + cells over total CD3 + cells in background sample.
Figure Legend Snippet: Mobilization of T cells is strain and TLR2-dependent. F1 (SJLxB6 wt ), F1 (SJLxB6 tlr− ) ( A , C ) or SJL (B, D ) mice were immunized with p139 in IFA in the presence of the indicated amounts of killed M tb ( A ), or of PPD ( B , C ) or of a 1∶1 w/w mixture of PAM2-(CSK)3 and PAM3-(CSK)3 ( D ). The number of mice for each group is indicated in the figure. Fourteen days later mice were sacrificed and the presence of T cells carrying the public TCR-beta chain in LN (closed symbols) and spleen (open symbols) was measured by immunoscope. Data are reported as RSI for the peak corresponding to the public BV10 TCR-beta chain for each individual mouse. Dashed lines indicate the cut off value for positivity (established as described in Results). E : 4 F1 (SJLxB6 wt , circles) mice and 5 F1 (SJLxB6 tlr2− , triangles) mice were immunized with regular CFA in PBS. Two weeks later cells from draining LN (full symbols) and spleen (open symbols) were recovered, labeled with CFSE and cultured in the presence or absence (background) of PPD. After 3 days, the number of CD3 + cells that had specifically divided in response to PPD was determined as % of CFSE low CD3 + cells over total CD3 + cells in the PPD stimulated sample minus % of CFSE low CD3 + cells over total CD3 + cells in background sample.

Techniques Used: Mouse Assay, Immunofluorescence, Labeling, Cell Culture

29) Product Images from "Xenotransplantation of interferon-gamma-pretreated clumps of a human mesenchymal stem cell/extracellular matrix complex induces mouse calvarial bone regeneration"

Article Title: Xenotransplantation of interferon-gamma-pretreated clumps of a human mesenchymal stem cell/extracellular matrix complex induces mouse calvarial bone regeneration

Journal: Stem Cell Research & Therapy

doi: 10.1186/s13287-017-0550-1

Phenotype profiles of C-MSC and C-MSCγ. Cell surface marker expression levels were monitored by flow cytometry, as described in the “ Methods ” section. The open histogram with blue or green lines indicates CD105-, CD90-, CD73-, CD34-, CD45-, HLA-DR-, or CD86-positive cells. The IgG control is shown with a solid histogram. CD cluster of differentiation, C-MSC clumps of a mesenchymal stem cell/extracellular matrix complex cultured in growth medium for 3 days, C-MSCγ (50) C-MSC stimulated with 50 ng/mL IFN-γ for 24 h before the end of the culture period, HLA human leukocyte antigens
Figure Legend Snippet: Phenotype profiles of C-MSC and C-MSCγ. Cell surface marker expression levels were monitored by flow cytometry, as described in the “ Methods ” section. The open histogram with blue or green lines indicates CD105-, CD90-, CD73-, CD34-, CD45-, HLA-DR-, or CD86-positive cells. The IgG control is shown with a solid histogram. CD cluster of differentiation, C-MSC clumps of a mesenchymal stem cell/extracellular matrix complex cultured in growth medium for 3 days, C-MSCγ (50) C-MSC stimulated with 50 ng/mL IFN-γ for 24 h before the end of the culture period, HLA human leukocyte antigens

Techniques Used: Marker, Expressing, Flow Cytometry, Cytometry, Cell Culture

30) Product Images from "DC targeting DNA vaccines induce protective and therapeutic antitumor immunity in mice"

Article Title: DC targeting DNA vaccines induce protective and therapeutic antitumor immunity in mice

Journal: International Journal of Clinical and Experimental Medicine

doi:

Plasmid DNA vaccines encoding secreted scFv N418 -HER2/neu fusion proteins. A. Schematic representation of expression vectors. scFv N418 -HER2, scFv N418 -neu, pcDNA3.1-HER2, or pcDNA3.1-neu encode under the control of a CMV promoter, all the fusion proteins consisting of an signal peptide, amino acid residues 1 to 222 of human HER2 or amino acid residues 1 to 224 of rat neu, and COOH-terminal Myc tag. The control plasmids pcDNA3.1-HER2 and pcDNA3.1-neu lack the scFv N418 domain. B. 293T cells grown in 100-mm dishes were transfected with various expression vectors using Lipofectamine 2000 (invitrogen). Immunoblot analysis of supernatants from 293T cells transfected with scFv N418 -HER2, scFv N418 -neu, pcDNA3.1-HER2 and pcDNA3.1-neu (lane 1, 2, 3 and 4). Vaccine proteins were probed with mouse anti-Myc tag mAb followed by HRP-conjugated secondary anti-mouse antibody. C. Binding of scFv N418 -HER2 fusion protein from culture supernatant of transfected 293T cells to mouse dendrtic cells was investigated by fluorescence-activated cell sorting analysis with mAb 9E10 followed by PE-conjugated secondary antibody. Control cells were treated with supernatant from mock-transfected cells.
Figure Legend Snippet: Plasmid DNA vaccines encoding secreted scFv N418 -HER2/neu fusion proteins. A. Schematic representation of expression vectors. scFv N418 -HER2, scFv N418 -neu, pcDNA3.1-HER2, or pcDNA3.1-neu encode under the control of a CMV promoter, all the fusion proteins consisting of an signal peptide, amino acid residues 1 to 222 of human HER2 or amino acid residues 1 to 224 of rat neu, and COOH-terminal Myc tag. The control plasmids pcDNA3.1-HER2 and pcDNA3.1-neu lack the scFv N418 domain. B. 293T cells grown in 100-mm dishes were transfected with various expression vectors using Lipofectamine 2000 (invitrogen). Immunoblot analysis of supernatants from 293T cells transfected with scFv N418 -HER2, scFv N418 -neu, pcDNA3.1-HER2 and pcDNA3.1-neu (lane 1, 2, 3 and 4). Vaccine proteins were probed with mouse anti-Myc tag mAb followed by HRP-conjugated secondary anti-mouse antibody. C. Binding of scFv N418 -HER2 fusion protein from culture supernatant of transfected 293T cells to mouse dendrtic cells was investigated by fluorescence-activated cell sorting analysis with mAb 9E10 followed by PE-conjugated secondary antibody. Control cells were treated with supernatant from mock-transfected cells.

Techniques Used: Plasmid Preparation, Expressing, Transfection, Binding Assay, Fluorescence, FACS

31) Product Images from "Temporal Regulation of Interleukin-12p70 (IL-12p70) and IL-12-Related Cytokines in Splenic Dendritic Cell Subsets during Leishmania donovani Infection ▿"

Article Title: Temporal Regulation of Interleukin-12p70 (IL-12p70) and IL-12-Related Cytokines in Splenic Dendritic Cell Subsets during Leishmania donovani Infection ▿

Journal: Infection and Immunity

doi: 10.1128/IAI.00643-07

CD11c + DX5 + DC are the major producers of IFN-γ. (A) CD11c hi DC subsets from naïve mice and mice infected with a low dose for 5 h were sorted on the basis of CD4 and CD8 expression, and ex vivo IFN-γ mRNA accumulation in sorted CD4 + , CD8 + , and DN DC was examined by real-time RT-PCR. Target genes were normalized against HPRT. Data represent the means plus standard errors of the means (error bars) from three separate experiments. (B) Splenic CD11c hi cells from mice infected for 5 h with L. donovani were enriched using CD11c microbeads. Enriched CD11c hi cells were then cultured in brefeldin A (10 μg/ml) for 4 h and surface stained with antibodies for CD11c, CD4, CD8, and CD49b (DX5). Fixed-cell samples were then permeabilized and stained for intracellular IFN-γ. Dot plots were gated on CD11c hi cells. Quadrant gates were set on isotype controls. The numbers indicate the percentage of events within the three quadrants. This figure shows the results of one experiment that was representative of three independent experiments.
Figure Legend Snippet: CD11c + DX5 + DC are the major producers of IFN-γ. (A) CD11c hi DC subsets from naïve mice and mice infected with a low dose for 5 h were sorted on the basis of CD4 and CD8 expression, and ex vivo IFN-γ mRNA accumulation in sorted CD4 + , CD8 + , and DN DC was examined by real-time RT-PCR. Target genes were normalized against HPRT. Data represent the means plus standard errors of the means (error bars) from three separate experiments. (B) Splenic CD11c hi cells from mice infected for 5 h with L. donovani were enriched using CD11c microbeads. Enriched CD11c hi cells were then cultured in brefeldin A (10 μg/ml) for 4 h and surface stained with antibodies for CD11c, CD4, CD8, and CD49b (DX5). Fixed-cell samples were then permeabilized and stained for intracellular IFN-γ. Dot plots were gated on CD11c hi cells. Quadrant gates were set on isotype controls. The numbers indicate the percentage of events within the three quadrants. This figure shows the results of one experiment that was representative of three independent experiments.

Techniques Used: Mouse Assay, Infection, Expressing, Ex Vivo, Quantitative RT-PCR, Cell Culture, Staining

32) Product Images from "Phage Antibodies Obtained by Competitive Selection on Complement-Resistant Moraxella (Branhamella) catarrhalis Recognize the High-Molecular-Weight Outer Membrane Protein"

Article Title: Phage Antibodies Obtained by Competitive Selection on Complement-Resistant Moraxella (Branhamella) catarrhalis Recognize the High-Molecular-Weight Outer Membrane Protein

Journal: Infection and Immunity

doi:

Flow cytometric detection of scFv binding to complement-resistant  M. catarrhalis . Binding of scFv to whole 9.21R bacteria was visualized by the Myc tag-specific MAb 9E10, followed by a fluorescein isothiocyanate-labeled goat anti-mouse antibody. Black graph, scFv B12; white graph, negative control scFv HM1.
Figure Legend Snippet: Flow cytometric detection of scFv binding to complement-resistant M. catarrhalis . Binding of scFv to whole 9.21R bacteria was visualized by the Myc tag-specific MAb 9E10, followed by a fluorescein isothiocyanate-labeled goat anti-mouse antibody. Black graph, scFv B12; white graph, negative control scFv HM1.

Techniques Used: Flow Cytometry, Binding Assay, Labeling, Negative Control

33) Product Images from "Failure of Oral Atorvastatin to Modulate a Murine Model of Systemic Lupus Erythematosus"

Article Title: Failure of Oral Atorvastatin to Modulate a Murine Model of Systemic Lupus Erythematosus

Journal: Arthritis and rheumatism

doi: 10.1002/art.23605

In vitro T cell assays. Splenic T cells from untreated and atorvastatin-treated NZB/NZW mice were stimulated with anti-CD3 and anti-CD28 antibodies, as described in Materials and Methods. A , Proliferation, as assessed by standard thymidine incorporation
Figure Legend Snippet: In vitro T cell assays. Splenic T cells from untreated and atorvastatin-treated NZB/NZW mice were stimulated with anti-CD3 and anti-CD28 antibodies, as described in Materials and Methods. A , Proliferation, as assessed by standard thymidine incorporation

Techniques Used: In Vitro, Mouse Assay

34) Product Images from "Indoleamine 2,3-Dioxygenase Controls Fungal Loads and Immunity in Paracoccidioidomicosis but is More Important to Susceptible than Resistant Hosts"

Article Title: Indoleamine 2,3-Dioxygenase Controls Fungal Loads and Immunity in Paracoccidioidomicosis but is More Important to Susceptible than Resistant Hosts

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0003330

IDO inhibition increases the migration of naïve and effector/memory CD4 + and CD8 + T cells to the lungs of A/J and B10.A mice, but reduces the number of regulatory T cells. ( A, B, C, D ) Characterization of CD4 + , CD8 + T cells by flow cytometry in the lung infiltrating leucocytes (LIL) from 1MT treated and untreated A/J and B10.A mice inoculated i.t. with 1×10 6 P. brasiliensis yeast cells. At weeks 2 and 8 after infection, lung cell suspensions were obtained and stained as described in Materials and Methods . The acquisition and analysis gates were restricted to lymphocytes. The total numbers of effector (CD44 high CD62L low ) and naïve (CD44 low CD62L high ) CD4 + and CD8 + T cells at weeks 2 and 8 after infection were determined. ( E, F ) To characterize the number of Treg cells in LIL at weeks 2 and 8 weeks of infection, cells positive for surface staining of CD25 and intracellular Foxp3 expression were back-gated on the CD4 + T cell population. The data represent the means ± SEM of 6–8 mice per group and are representative of three independent experiments (* p
Figure Legend Snippet: IDO inhibition increases the migration of naïve and effector/memory CD4 + and CD8 + T cells to the lungs of A/J and B10.A mice, but reduces the number of regulatory T cells. ( A, B, C, D ) Characterization of CD4 + , CD8 + T cells by flow cytometry in the lung infiltrating leucocytes (LIL) from 1MT treated and untreated A/J and B10.A mice inoculated i.t. with 1×10 6 P. brasiliensis yeast cells. At weeks 2 and 8 after infection, lung cell suspensions were obtained and stained as described in Materials and Methods . The acquisition and analysis gates were restricted to lymphocytes. The total numbers of effector (CD44 high CD62L low ) and naïve (CD44 low CD62L high ) CD4 + and CD8 + T cells at weeks 2 and 8 after infection were determined. ( E, F ) To characterize the number of Treg cells in LIL at weeks 2 and 8 weeks of infection, cells positive for surface staining of CD25 and intracellular Foxp3 expression were back-gated on the CD4 + T cell population. The data represent the means ± SEM of 6–8 mice per group and are representative of three independent experiments (* p

Techniques Used: Inhibition, Migration, Mouse Assay, Flow Cytometry, Cytometry, Infection, Staining, Expressing

IDO blockade increases the presence of IL-17 + , but reduces the number of IFN-γ + T cells in the lungs of A/J and B10.A mice. Lymphocytes from lung infiltrating leukocytes (LIL) obtained from groups of 5–6 mice were gated based on forward/side scatters analysis. Gated cells were assessed for CD4 + , CD8 + , γδ + and NKT + markers using labeled antibodies. The presence of IL-17 + , IFN-γ + and IL-4 + T cells in the LIL was determined by intracellular cytokine staining at week 2 after infection. Lung cells were stimulated in vitro with PMA/ionomycin for 6 h, the last 4 h in the presence of brefeldin A, and subjected to intracellular staining for IL-17, IL-4 and IFN-γ. The results are representative of three experiments with equivalent results (* p
Figure Legend Snippet: IDO blockade increases the presence of IL-17 + , but reduces the number of IFN-γ + T cells in the lungs of A/J and B10.A mice. Lymphocytes from lung infiltrating leukocytes (LIL) obtained from groups of 5–6 mice were gated based on forward/side scatters analysis. Gated cells were assessed for CD4 + , CD8 + , γδ + and NKT + markers using labeled antibodies. The presence of IL-17 + , IFN-γ + and IL-4 + T cells in the LIL was determined by intracellular cytokine staining at week 2 after infection. Lung cells were stimulated in vitro with PMA/ionomycin for 6 h, the last 4 h in the presence of brefeldin A, and subjected to intracellular staining for IL-17, IL-4 and IFN-γ. The results are representative of three experiments with equivalent results (* p

Techniques Used: Mouse Assay, Labeling, Staining, Infection, In Vitro

IDO inhibition increases the influx of myeloid (CD11c high CD11b + ), lymphoid (CD11c high CD8 + ) and plasmacytoid (CD11c high B220 + ) dendritic cells to the lungs of A/J and B10.A mice. 1MT treated or untreated B10.A and A/J mice were infected i.t. with 1×10 6 yeast cells of P. brasiliensis , and at 2 ( A ) and 8 ( B ) weeks after infection, lung inflammatory cells were obtained and lung DC subsets characterized using specific antibodies. Cell phenotypes were analyzed by flow cytometry as described in Materials and Methods . The data represent means ± SEM of 5–7 mice per group and are representative of three independent experiments (* p
Figure Legend Snippet: IDO inhibition increases the influx of myeloid (CD11c high CD11b + ), lymphoid (CD11c high CD8 + ) and plasmacytoid (CD11c high B220 + ) dendritic cells to the lungs of A/J and B10.A mice. 1MT treated or untreated B10.A and A/J mice were infected i.t. with 1×10 6 yeast cells of P. brasiliensis , and at 2 ( A ) and 8 ( B ) weeks after infection, lung inflammatory cells were obtained and lung DC subsets characterized using specific antibodies. Cell phenotypes were analyzed by flow cytometry as described in Materials and Methods . The data represent means ± SEM of 5–7 mice per group and are representative of three independent experiments (* p

Techniques Used: Inhibition, Mouse Assay, Infection, Flow Cytometry, Cytometry

35) Product Images from "Identification of novel myeloma-specific XBP1 peptides able to generate cytotoxic T lymphocytes: A potential therapeutic application in multiple myeloma"

Article Title: Identification of novel myeloma-specific XBP1 peptides able to generate cytotoxic T lymphocytes: A potential therapeutic application in multiple myeloma

Journal: Leukemia : official journal of the Leukemia Society of America, Leukemia Research Fund, U.K

doi: 10.1038/leu.2011.120

Identification of HLA-A2-specific XBP1 peptides and improvement of MHC binding stability by peptide modification Figure 1a. HLA-A2 binding affinity of non-spliced XBP1 peptides. T2 cells were pulsed overnight with respective XBP1 peptide (50 µg/ml) in serum-fee media. Influenza virus matrix protein 58–66 was used as the positive control and T2 cell in media alone as baseline controls in these experiments. Following incubation, T2 cells were harvested, washed, and stained with HLA-A2-FITC mAb for flow cytometric analysis. HLA-A2 binding is shown as an increase in HLA-A2 mean fluorescence intensity (MFI). XBP1 184–192 (NISPWILAV) showed the highest HLA-A2 binding affinity among the non-spliced XBP1 peptides. The values represent the mean ± SE of three separate experiments. Figure 1b. HLA-A2 binding stability of non-spliced XBP1 peptides. Native or heteroclitic XBP1 peptide (50 µg/ml)-pulsed T2 cells were washed and incubated with Brefeldin A. At 0, 2, 4, 6, and 18 hours incubation, the cells were stained with HLA-A2-FITC mAb for flow cytometric analysis. Heteroclitic XBP1 184–192 ( Y ISPWILAV) peptide showed increased HLA-A2 binding stability compared to the native XBP1 184–192 (NISPWILAV) peptide. Binding stability of the heteroclitic peptide was higher than influenza virus matrix protein 58–66 (GILGFVFTL), which was used as the HLA-A2-specific positive control peptide. The values represent the mean ± SE of three separate experiments. Figure 1c. HLA-A2 binding affinity of spliced XBP1 peptides. Spliced XBP1 peptides were evaluated for their HLA-A2 binding affinity as described in Figure 1a . XBP1 SP 196–204 (GILDNLDPV) and XBP1 SP 367–375 (ELFPQLISV) showed the highest HLA-A2 binding affinity among the spliced XBP1 peptides. The values represent the mean ± SE of three separate experiments. Figure 1d. HLA-A2 binding stability of spliced XBP1 peptides. Spliced XBP1 peptides were analyzed for their HLA-A2 binding stability as discussed in Figure 1b . Heteroclitic XBP1 SP 367–375 ( Y LFPQLISV) peptide displayed increased HLA-A2 binding stability compared to its native XBP1 SP 367–375 (ELFPQLISV) or another native XBP1 SP 196–204 (GILDNLDPV) peptide. The values represent the mean ± SE of three separate experiments.
Figure Legend Snippet: Identification of HLA-A2-specific XBP1 peptides and improvement of MHC binding stability by peptide modification Figure 1a. HLA-A2 binding affinity of non-spliced XBP1 peptides. T2 cells were pulsed overnight with respective XBP1 peptide (50 µg/ml) in serum-fee media. Influenza virus matrix protein 58–66 was used as the positive control and T2 cell in media alone as baseline controls in these experiments. Following incubation, T2 cells were harvested, washed, and stained with HLA-A2-FITC mAb for flow cytometric analysis. HLA-A2 binding is shown as an increase in HLA-A2 mean fluorescence intensity (MFI). XBP1 184–192 (NISPWILAV) showed the highest HLA-A2 binding affinity among the non-spliced XBP1 peptides. The values represent the mean ± SE of three separate experiments. Figure 1b. HLA-A2 binding stability of non-spliced XBP1 peptides. Native or heteroclitic XBP1 peptide (50 µg/ml)-pulsed T2 cells were washed and incubated with Brefeldin A. At 0, 2, 4, 6, and 18 hours incubation, the cells were stained with HLA-A2-FITC mAb for flow cytometric analysis. Heteroclitic XBP1 184–192 ( Y ISPWILAV) peptide showed increased HLA-A2 binding stability compared to the native XBP1 184–192 (NISPWILAV) peptide. Binding stability of the heteroclitic peptide was higher than influenza virus matrix protein 58–66 (GILGFVFTL), which was used as the HLA-A2-specific positive control peptide. The values represent the mean ± SE of three separate experiments. Figure 1c. HLA-A2 binding affinity of spliced XBP1 peptides. Spliced XBP1 peptides were evaluated for their HLA-A2 binding affinity as described in Figure 1a . XBP1 SP 196–204 (GILDNLDPV) and XBP1 SP 367–375 (ELFPQLISV) showed the highest HLA-A2 binding affinity among the spliced XBP1 peptides. The values represent the mean ± SE of three separate experiments. Figure 1d. HLA-A2 binding stability of spliced XBP1 peptides. Spliced XBP1 peptides were analyzed for their HLA-A2 binding stability as discussed in Figure 1b . Heteroclitic XBP1 SP 367–375 ( Y LFPQLISV) peptide displayed increased HLA-A2 binding stability compared to its native XBP1 SP 367–375 (ELFPQLISV) or another native XBP1 SP 196–204 (GILDNLDPV) peptide. The values represent the mean ± SE of three separate experiments.

Techniques Used: Binding Assay, Modification, Positive Control, Incubation, Staining, Flow Cytometry, Fluorescence

36) Product Images from "Reduction of complement factor H binding to CLL cells improves the induction of rituximab-mediated complement-dependent cytotoxicity"

Article Title: Reduction of complement factor H binding to CLL cells improves the induction of rituximab-mediated complement-dependent cytotoxicity

Journal: Leukemia

doi: 10.1038/leu.2013.169

Effects of hSCR18–20, HD1A and MEM43 on RTX-induced CDC of primary CLL cells administered alone or in combination. ( a ) Patient cells were treated with RTX in the absence or presence of blocking anti-CD55 (HD1A), blocking anti-CD59 (MEM43) or hSCR18–20 under standard conditions. Addition of HD1A and hSCR18–20 significantly enhanced RTX-induced CDC (29% and 24%, respectively), whereas the blocking of CD59 by MEM43 showed minor improvement (10%). Combination of HD1A and hSCR18–20 resulted in significantly enhanced effects as compared with the effects of each compound individually. Survival rates were calculated according to the hiNHS control, which defined 100% survival. Error bars: s.e.m, n =20. ( b ) Treatment of primary CLL cells with HD1A, MEM43 or hSCR18–20 and active complement in the absence of RTX did not induce CDC (gray bars), as compared with the NHS control (white bar, n =16). ( c ) Improvement of RTX-induced CDC by blocking anti-CD55 (HD1A) was significant ( P =0.0019) in comparison to non-blocking anti-CD55 ( n =7). ( d ) No significant difference between the blocking anti-CD59 (MEM43) and non-blocking anti-CD59 was observed ( n =6). Survival rates were calculated according to the hiNHS control, which defined 100% survival. Error bars: s.e.m.
Figure Legend Snippet: Effects of hSCR18–20, HD1A and MEM43 on RTX-induced CDC of primary CLL cells administered alone or in combination. ( a ) Patient cells were treated with RTX in the absence or presence of blocking anti-CD55 (HD1A), blocking anti-CD59 (MEM43) or hSCR18–20 under standard conditions. Addition of HD1A and hSCR18–20 significantly enhanced RTX-induced CDC (29% and 24%, respectively), whereas the blocking of CD59 by MEM43 showed minor improvement (10%). Combination of HD1A and hSCR18–20 resulted in significantly enhanced effects as compared with the effects of each compound individually. Survival rates were calculated according to the hiNHS control, which defined 100% survival. Error bars: s.e.m, n =20. ( b ) Treatment of primary CLL cells with HD1A, MEM43 or hSCR18–20 and active complement in the absence of RTX did not induce CDC (gray bars), as compared with the NHS control (white bar, n =16). ( c ) Improvement of RTX-induced CDC by blocking anti-CD55 (HD1A) was significant ( P =0.0019) in comparison to non-blocking anti-CD55 ( n =7). ( d ) No significant difference between the blocking anti-CD59 (MEM43) and non-blocking anti-CD59 was observed ( n =6). Survival rates were calculated according to the hiNHS control, which defined 100% survival. Error bars: s.e.m.

Techniques Used: Blocking Assay

37) Product Images from "Inactivation of nuclear GSK3β by Ser389 phosphorylation promotes lymphocyte fitness during DNA double-strand break response"

Article Title: Inactivation of nuclear GSK3β by Ser389 phosphorylation promotes lymphocyte fitness during DNA double-strand break response

Journal: Nature Communications

doi: 10.1038/ncomms10553

Inactivation of GSK3β by p38 MAPK is specifically induced by DSBs. ( a ) Western blot analysis of P-S 389 GSK3β, P-S 9 GSK3β, GSK3β, P-p38 MAPK and p38 MAPK in total thymocytes (T), double positive thymocytes (DP), double negative thymocytes (DN), mature CD4 T cells and mature CD8 T cells. Actin is shown as a loading control. ( b ) Mouse CD4 splenocytes were treated with media alone (Med) or stimulated with anti-CD3 and anti-CD28 Abs for 18 h and the levels of P-S 389 GSK3β, GSK3β, P-p38 MAPK and p38 MAPK were determined by western blotting. Thymocytes are included as a positive control. ( c ) Mouse CD4 splenocytes were treated with media alone (Med) or doxorubicin (Dox) for 18 h and the levels of P-S 389 GSK3β, total GSK3β, P-p38 MAPK and total p38 MAPK were determined by western blotting. ( d ) Mouse CD4 splenocytes were treated with media alone (Med) or doxorubicin (Dox) for 18 h. Levels of P-S 389 GSK3β, total GSK3β and γH2AX were determined by western blotting. GAPDH is shown as a loading control. ( e ) Mouse CD4 splenocytes were treated with doxorubicin for 18 h and cell death was measured by AnnexinV staining and flow cytometry ( n =3, ±s.e.m.). ( f ) Human CD4 cells were treated with media alone (Med) or doxorubicin (Dox) for 18 h. P-T 390 GSK3β, total GSK3β and γH2AX were examined by western blotting. ( g ) WT and T-cell-specific p38α conditional knockout (cKO) mice were left unexposed or exposed to 4 Gy of X-rays. After 1.5 h, CD4 splenocytes were isolated and P-S 389 GSK3β and total GSK3β assessed by western blotting. Actin is shown as a loading control. ( h ) Relative levels of total GSK3β, P-T 390 GSK3β and the relative ratio of P-T 390 GSK3β to total GSK3β at baseline (BL) and after radiotherapy (Rad) in CD4 cells from breast cancer patients, determined by the Odyssey system ( n =4). ( i ) Relative ratio of P-T 390 GSK3β to total GSK3β in CD4 cells from healthy donors isolated in two different days (baseline 1 and 2) for each subject ( n =3). * P value
Figure Legend Snippet: Inactivation of GSK3β by p38 MAPK is specifically induced by DSBs. ( a ) Western blot analysis of P-S 389 GSK3β, P-S 9 GSK3β, GSK3β, P-p38 MAPK and p38 MAPK in total thymocytes (T), double positive thymocytes (DP), double negative thymocytes (DN), mature CD4 T cells and mature CD8 T cells. Actin is shown as a loading control. ( b ) Mouse CD4 splenocytes were treated with media alone (Med) or stimulated with anti-CD3 and anti-CD28 Abs for 18 h and the levels of P-S 389 GSK3β, GSK3β, P-p38 MAPK and p38 MAPK were determined by western blotting. Thymocytes are included as a positive control. ( c ) Mouse CD4 splenocytes were treated with media alone (Med) or doxorubicin (Dox) for 18 h and the levels of P-S 389 GSK3β, total GSK3β, P-p38 MAPK and total p38 MAPK were determined by western blotting. ( d ) Mouse CD4 splenocytes were treated with media alone (Med) or doxorubicin (Dox) for 18 h. Levels of P-S 389 GSK3β, total GSK3β and γH2AX were determined by western blotting. GAPDH is shown as a loading control. ( e ) Mouse CD4 splenocytes were treated with doxorubicin for 18 h and cell death was measured by AnnexinV staining and flow cytometry ( n =3, ±s.e.m.). ( f ) Human CD4 cells were treated with media alone (Med) or doxorubicin (Dox) for 18 h. P-T 390 GSK3β, total GSK3β and γH2AX were examined by western blotting. ( g ) WT and T-cell-specific p38α conditional knockout (cKO) mice were left unexposed or exposed to 4 Gy of X-rays. After 1.5 h, CD4 splenocytes were isolated and P-S 389 GSK3β and total GSK3β assessed by western blotting. Actin is shown as a loading control. ( h ) Relative levels of total GSK3β, P-T 390 GSK3β and the relative ratio of P-T 390 GSK3β to total GSK3β at baseline (BL) and after radiotherapy (Rad) in CD4 cells from breast cancer patients, determined by the Odyssey system ( n =4). ( i ) Relative ratio of P-T 390 GSK3β to total GSK3β in CD4 cells from healthy donors isolated in two different days (baseline 1 and 2) for each subject ( n =3). * P value

Techniques Used: Western Blot, Positive Control, Staining, Flow Cytometry, Cytometry, Knock-Out, Mouse Assay, Isolation

38) Product Images from "A dual role for the immune response in a mouse model of inflammation-associated lung cancer"

Article Title: A dual role for the immune response in a mouse model of inflammation-associated lung cancer

Journal: The Journal of Clinical Investigation

doi: 10.1172/JCI44796

BALB/c TKO lung tumors develop in the context of pulmonary and systemic inflammation. ( A ) Comparison of total leukocytes recovered from BAL fluid from 3-month-old BALB/c TKO and WT mice (left panel) and analysis of lymphoid and myeloid subtypes (middle panel) using flow cytometry. Right panel: Total spleen cells from the same WT and TKO mice are presented for comparison. ( B ) Quantification of inflammatory cytokines present in the BAL fluid (left panel) and serum (right panel) of 3-month-old BALB/c TKO and WT mice; each point represents an individual animal. Cytokines were measured using fluorescent anti-cytokine beads; IL-6 levels in the BAL fluid were confirmed by ELISA. Results represent an analysis of samples collected from 3 independent experiments. ( C and D ) Quantification of serum cytokines in aged BALB/c WT and TKO mice by ELISA. ( C ) Points represent individual animals. ( D ) Serum IL-17 in 1-year-old WT ( n = 6) and TKO ( n = 4) mice. ( E and F ) Cytokine production from 1 × 10 6 CD4 + T cells ( E ) stimulated for 48 hours using anti-CD3 (10 μg/ml) and anti-CD28 (2 μg/ml), or from CD11b + macrophages ( F ) cultured for 18 hours. Cells were isolated from spleens of 6- to 8-week-old mice by positive selection with magnetic beads (Miltenyi Biotec). Cytokines/chemokines were measured using anti-cytokine beads. Results are combined from 2 independent experiments with a total of 6 ( E ) or 4 ( F ) mice per group. Error bars represent SEM.
Figure Legend Snippet: BALB/c TKO lung tumors develop in the context of pulmonary and systemic inflammation. ( A ) Comparison of total leukocytes recovered from BAL fluid from 3-month-old BALB/c TKO and WT mice (left panel) and analysis of lymphoid and myeloid subtypes (middle panel) using flow cytometry. Right panel: Total spleen cells from the same WT and TKO mice are presented for comparison. ( B ) Quantification of inflammatory cytokines present in the BAL fluid (left panel) and serum (right panel) of 3-month-old BALB/c TKO and WT mice; each point represents an individual animal. Cytokines were measured using fluorescent anti-cytokine beads; IL-6 levels in the BAL fluid were confirmed by ELISA. Results represent an analysis of samples collected from 3 independent experiments. ( C and D ) Quantification of serum cytokines in aged BALB/c WT and TKO mice by ELISA. ( C ) Points represent individual animals. ( D ) Serum IL-17 in 1-year-old WT ( n = 6) and TKO ( n = 4) mice. ( E and F ) Cytokine production from 1 × 10 6 CD4 + T cells ( E ) stimulated for 48 hours using anti-CD3 (10 μg/ml) and anti-CD28 (2 μg/ml), or from CD11b + macrophages ( F ) cultured for 18 hours. Cells were isolated from spleens of 6- to 8-week-old mice by positive selection with magnetic beads (Miltenyi Biotec). Cytokines/chemokines were measured using anti-cytokine beads. Results are combined from 2 independent experiments with a total of 6 ( E ) or 4 ( F ) mice per group. Error bars represent SEM.

Techniques Used: Mouse Assay, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay, Cell Culture, Isolation, Selection, Magnetic Beads

39) Product Images from "Isolation of dendritic cells from umbilical cord blood using magnetic activated cell sorting or adherence"

Article Title: Isolation of dendritic cells from umbilical cord blood using magnetic activated cell sorting or adherence

Journal: Oncology Letters

doi: 10.3892/ol.2015.3198

Flow cytometric analysis of the dendritic cells on the 7th day of culture. (A–C) Cells that were cultured by the adherent method and (D–F) cells that were cultured by the magnetic activated cell sorting method. The cells in the gated population were further analyzed and identified to be (B and E) CD11C + /HLA-DR + and (C and F) CD11C + /CD80 + . CD, cluster of differentiation; HLA, human leukocyte antigen; APC, antigen-presenting cell.
Figure Legend Snippet: Flow cytometric analysis of the dendritic cells on the 7th day of culture. (A–C) Cells that were cultured by the adherent method and (D–F) cells that were cultured by the magnetic activated cell sorting method. The cells in the gated population were further analyzed and identified to be (B and E) CD11C + /HLA-DR + and (C and F) CD11C + /CD80 + . CD, cluster of differentiation; HLA, human leukocyte antigen; APC, antigen-presenting cell.

Techniques Used: Flow Cytometry, Cell Culture, FACS

40) Product Images from "Radioprotective effects of an acidic polysaccharide of Panax ginseng on bone marrow cells"

Article Title: Radioprotective effects of an acidic polysaccharide of Panax ginseng on bone marrow cells

Journal: Journal of Veterinary Science

doi: 10.4142/jvs.2007.8.1.39

APG significantly increased the number of BMs and DC progenitor cells in vivo . The mice were injected with PBS alone or APG, and then irradiated. After 4 days, the number of BMs obtained from PBS-injected (control) or APG-injected mice was counted (A) and the cells were cultured in the presence of GM-CSF to verify the presence of DC progenitor cells. The number of the dot plot indicates the percentage of CD11c and MHC II double positive cells (B). Result is a representative of 2 individual experiments. All values are represented as mean ± SD and indicated statistical significance *** p
Figure Legend Snippet: APG significantly increased the number of BMs and DC progenitor cells in vivo . The mice were injected with PBS alone or APG, and then irradiated. After 4 days, the number of BMs obtained from PBS-injected (control) or APG-injected mice was counted (A) and the cells were cultured in the presence of GM-CSF to verify the presence of DC progenitor cells. The number of the dot plot indicates the percentage of CD11c and MHC II double positive cells (B). Result is a representative of 2 individual experiments. All values are represented as mean ± SD and indicated statistical significance *** p

Techniques Used: In Vivo, Mouse Assay, Injection, Irradiation, Cell Culture

Related Articles

Flow Cytometry:

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Article Snippet: .. Briefly, beads with defined antibody binding capacity were incubated with cmHsp70.1-FITC mAb and analysed on a BD Biosciences FACSCalibur™ flow cytometer to obtain a standard curve. .. The capacity of cells to bind cmHsp70.1-FITC is presented as mean equivalents of soluble fluorochrome (MESF) divided by the antibody binding capacity (ABC).

Article Title: IL-21 Regulates the Differentiation of a Human ?? T Cell Subset Equipped with B Cell Helper Activity
Article Snippet: .. Vγ9Vδ2 cells were incubated in U-bottom 96-well plates with labelled mAbs in PBS containing 1% FCS, for 30 min at 4°C according to manufacturers’ recommendations, washed, and analyzed by flow cytometry on an FACSCalibur or FACSCanto II (BD Biosciences) and analyzed with FlowJo software (Tree Star). ..

Isolation:

Article Title: Molecular Profiling of Single Sca-1+/CD34+,− Cells--The Putative Murine Lung Stem Cells
Article Snippet: .. For initial molecular characterization of isolated cells, PCR on transcripts of Sca-1 , CD34 , CD45 and CD31 were performed. .. In order to differentiate between a more epithelial or mesenchymal phenotype of isolated cells, we conducted further PCRs specific for epithelial markers Epcam (Epithelial cell adhesion molecule), Itga (Integrin alpha-6) and Sftpc (Surfactant protein C) and mesenchymal markers CD90 (Thy-1) and Pdgfrα (platelet derived growth factor receptor alpha, CD140a), as suggested by McQualter et al. .

Cytometry:

Article Title: Immunotherapeutic Targeting of Membrane Hsp70-Expressing Tumors Using Recombinant Human Granzyme B
Article Snippet: .. Briefly, beads with defined antibody binding capacity were incubated with cmHsp70.1-FITC mAb and analysed on a BD Biosciences FACSCalibur™ flow cytometer to obtain a standard curve. .. The capacity of cells to bind cmHsp70.1-FITC is presented as mean equivalents of soluble fluorochrome (MESF) divided by the antibody binding capacity (ABC).

Article Title: IL-21 Regulates the Differentiation of a Human ?? T Cell Subset Equipped with B Cell Helper Activity
Article Snippet: .. Vγ9Vδ2 cells were incubated in U-bottom 96-well plates with labelled mAbs in PBS containing 1% FCS, for 30 min at 4°C according to manufacturers’ recommendations, washed, and analyzed by flow cytometry on an FACSCalibur or FACSCanto II (BD Biosciences) and analyzed with FlowJo software (Tree Star). ..

Polymerase Chain Reaction:

Article Title: Molecular Profiling of Single Sca-1+/CD34+,− Cells--The Putative Murine Lung Stem Cells
Article Snippet: .. For initial molecular characterization of isolated cells, PCR on transcripts of Sca-1 , CD34 , CD45 and CD31 were performed. .. In order to differentiate between a more epithelial or mesenchymal phenotype of isolated cells, we conducted further PCRs specific for epithelial markers Epcam (Epithelial cell adhesion molecule), Itga (Integrin alpha-6) and Sftpc (Surfactant protein C) and mesenchymal markers CD90 (Thy-1) and Pdgfrα (platelet derived growth factor receptor alpha, CD140a), as suggested by McQualter et al. .

Incubation:

Article Title: Immunotherapeutic Targeting of Membrane Hsp70-Expressing Tumors Using Recombinant Human Granzyme B
Article Snippet: .. Briefly, beads with defined antibody binding capacity were incubated with cmHsp70.1-FITC mAb and analysed on a BD Biosciences FACSCalibur™ flow cytometer to obtain a standard curve. .. The capacity of cells to bind cmHsp70.1-FITC is presented as mean equivalents of soluble fluorochrome (MESF) divided by the antibody binding capacity (ABC).

Article Title: IL-21 Regulates the Differentiation of a Human ?? T Cell Subset Equipped with B Cell Helper Activity
Article Snippet: .. Vγ9Vδ2 cells were incubated in U-bottom 96-well plates with labelled mAbs in PBS containing 1% FCS, for 30 min at 4°C according to manufacturers’ recommendations, washed, and analyzed by flow cytometry on an FACSCalibur or FACSCanto II (BD Biosciences) and analyzed with FlowJo software (Tree Star). ..

other:

Article Title: STIM1 deficiency is linked to Alzheimer’s disease and triggers cell death in SH-SY5Y cells by upregulation of L-type voltage-operated Ca2+ entry
Article Snippet: Antibodies The rabbit polyclonal anti-STIM1 antibody (raised against the C-terminus) was from ProSci Inc. (Poway, CA, USA), and the mouse monoclonal anti-STIM1 antibody (raised against the N-terminus) was from BD Biosciences (Franklin Lakes, NJ, USA); the mouse monoclonal anti-tubulin beta 3, class III (TUBB3), and the mouse anti-beta tubulin (clone TUB 2.1) were from Sigma-Aldrich (St. Louis, MO, USA); the mouse monoclonal p21 antibody (p21CIP1) and the mouse monoclonal anti-GAPDH antibody were from Santa Cruz Biotechnology (Heidelberg, Germany).

Marker:

Article Title: Mbd2 enables tumourigenesis within the intestine while preventing tumour‐promoting inflammation
Article Snippet: .. To identify cells which had lost Apc , we used nuclear β‐catenin as a surrogate marker, using a mouse monoclonal anti‐β‐catenin antibody (Cat No 610154; BD Biosciences, Wokingham, UK) at 1:200. .. For Paneth cell detection, we used a rabbit polyclonal anti‐lysozyme antibody (Cat No RB‐372; Neomarkers/Labvision, Fisher Scientific) at 1:200.

Staining:

Article Title: Local CD4 and CD8 T-Cell Reactivity to HSV-1 Antigens Documents Broad Viral Protein Expression and Immune Competence in Latently Infected Human Trigeminal Ganglia
Article Snippet: .. Cells were harvested, stained for CD3 (BD), CD8 (BD) and CD137 (Miltenyi). .. Cells that co-expressed CD3, CD8, and CD137 were enriched with a BD FACS Aria cell sorter, expanded by PHA stimulation and used in HSV-1 ORFeome screens as described , .

Binding Assay:

Article Title: Immunotherapeutic Targeting of Membrane Hsp70-Expressing Tumors Using Recombinant Human Granzyme B
Article Snippet: .. Briefly, beads with defined antibody binding capacity were incubated with cmHsp70.1-FITC mAb and analysed on a BD Biosciences FACSCalibur™ flow cytometer to obtain a standard curve. .. The capacity of cells to bind cmHsp70.1-FITC is presented as mean equivalents of soluble fluorochrome (MESF) divided by the antibody binding capacity (ABC).

Software:

Article Title: IL-21 Regulates the Differentiation of a Human ?? T Cell Subset Equipped with B Cell Helper Activity
Article Snippet: .. Vγ9Vδ2 cells were incubated in U-bottom 96-well plates with labelled mAbs in PBS containing 1% FCS, for 30 min at 4°C according to manufacturers’ recommendations, washed, and analyzed by flow cytometry on an FACSCalibur or FACSCanto II (BD Biosciences) and analyzed with FlowJo software (Tree Star). ..

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    Becton Dickinson rat monoclonal anti mouse syndecan 4
    ATXβ controls breast cancer cell metastasis through an SDC4-dependent mechanism ( A ) 4T1 cell adhesion to increasing amounts of ATXβ, BSA (400 ng) was used as control (left panels). Data represent the mean of adherent cells/mm 2 ±SD of adherent cells of 3 experiments performed in 8 replicates (right panel). ( B ) Flow cytometry detection of cell surface expression of <t>syndecan-4</t> (SDC4) in 4T1 cells. Cells were immunostained with KY/8.2 monoclonal antibody (anti-SDC4) (black bar), or isotype control antibody MOPC21 (grey bar). NT: not treated cells (open bar). ( C ) Inhibition of 4T1 cell adhesion on ATXβ with KY/8.2 antibody (anti-SDC4). Indicated cell lines were preincubated for 1 h in the presence of KY/8.2 or MOPC21 antibodies (10 µg/mL). Data represent the mean of adherent cells/mm 2 ±SD of adherent cells of 3 experiments performed in 8 replicates ( *** : P
    Rat Monoclonal Anti Mouse Syndecan 4, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson plate bound anti cd3 mab
    Egg deposition within PP disrupts lymphoid microarchitecture and causes loss of cellularity. (A) DsRed and eYFP expression demarcate B cell follicles and inter-follicular T cell zones of PP. Images are 3D rendered multi-photon confocal display of excised PP tissue derived from VaDsRed/CD19+ eYFP double fluorescent reporter mice (red = <t>CD3+</t> cells; green = CD19+ cells; blue = second-harmonic generation by collagen fibres). Auto-fluorescent egg (arrows) proximal to PP at 6 and 8 weeks pi. All data are representative of two independent experiments, n = 3 mice. (B) Stereo fluorescent stereomicroscope images of PP in naïve, and infected CD3+/CD19+ reporter mice. Auto-fluorescent eggs (arrows) co-localise to T and B cell zones of PP at 14 weeks (lower panel). (C) 3D multiphoton confocal image of PP egg-granuloma (enlargement of section inset shown in B as dotted line); Sm = autofluorescent egg; arrows = cellular infiltrate; blue = second-harmonic generation by collagen fibres. (D) Numbers of CD11b+ cells, CD11b+SigLecF-F4/80+ macrophages, and CD11b+SigLecF+F4/80lo eosinophils in PP cell suspensions enumerated by flow cytometry in naïve mice, or after infection. (E) CD19+ PP follicle area in double reporter mice (min 3 PP/mouse). (F) Total viable PP, B220+ and IgA+B220+ B cell yields/mouse and (G) proportion of B220+ cells of total PP. (H) Stereo fluorescent stereomicroscope images of mLN in situ from naïve and infected CD3+ (red)/CD19+ (green) reporter mice. (I) mLN follicle area, (J) total viable cell and B220+ B cell yields in the mLN of naïve and infected CD3+T cell/CD19+B cell double reporter mice. All data; *P
    Plate Bound Anti Cd3 Mab, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 90/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Becton Dickinson mouse monoclonal anti cd45 igg antibody
    Phenotype profiles of C-MSC and C-MSCγ. Cell surface marker expression levels were monitored by flow cytometry, as described in the “ Methods ” section. The open histogram with blue or green lines indicates CD105-, CD90-, CD73-, CD34-, <t>CD45-,</t> HLA-DR-, or CD86-positive cells. The <t>IgG</t> control is shown with a solid histogram. CD cluster of differentiation, C-MSC clumps of a mesenchymal stem cell/extracellular matrix complex cultured in growth medium for 3 days, C-MSCγ (50) C-MSC stimulated with 50 ng/mL IFN-γ for 24 h before the end of the culture period, HLA human leukocyte antigens
    Mouse Monoclonal Anti Cd45 Igg Antibody, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 91/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse monoclonal anti cd45 igg antibody/product/Becton Dickinson
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    Image Search Results


    ATXβ controls breast cancer cell metastasis through an SDC4-dependent mechanism ( A ) 4T1 cell adhesion to increasing amounts of ATXβ, BSA (400 ng) was used as control (left panels). Data represent the mean of adherent cells/mm 2 ±SD of adherent cells of 3 experiments performed in 8 replicates (right panel). ( B ) Flow cytometry detection of cell surface expression of syndecan-4 (SDC4) in 4T1 cells. Cells were immunostained with KY/8.2 monoclonal antibody (anti-SDC4) (black bar), or isotype control antibody MOPC21 (grey bar). NT: not treated cells (open bar). ( C ) Inhibition of 4T1 cell adhesion on ATXβ with KY/8.2 antibody (anti-SDC4). Indicated cell lines were preincubated for 1 h in the presence of KY/8.2 or MOPC21 antibodies (10 µg/mL). Data represent the mean of adherent cells/mm 2 ±SD of adherent cells of 3 experiments performed in 8 replicates ( *** : P

    Journal: Oncotarget

    Article Title: Autotaxin-β interaction with the cell surface via syndecan-4 impacts on cancer cell proliferation and metastasis

    doi: 10.18632/oncotarget.26039

    Figure Lengend Snippet: ATXβ controls breast cancer cell metastasis through an SDC4-dependent mechanism ( A ) 4T1 cell adhesion to increasing amounts of ATXβ, BSA (400 ng) was used as control (left panels). Data represent the mean of adherent cells/mm 2 ±SD of adherent cells of 3 experiments performed in 8 replicates (right panel). ( B ) Flow cytometry detection of cell surface expression of syndecan-4 (SDC4) in 4T1 cells. Cells were immunostained with KY/8.2 monoclonal antibody (anti-SDC4) (black bar), or isotype control antibody MOPC21 (grey bar). NT: not treated cells (open bar). ( C ) Inhibition of 4T1 cell adhesion on ATXβ with KY/8.2 antibody (anti-SDC4). Indicated cell lines were preincubated for 1 h in the presence of KY/8.2 or MOPC21 antibodies (10 µg/mL). Data represent the mean of adherent cells/mm 2 ±SD of adherent cells of 3 experiments performed in 8 replicates ( *** : P

    Article Snippet: Rat monoclonal anti-mouse syndecan-4 (KY/8.2) was from Becton Dickinson Biosciences (Franklin Lakes, NJ, USA) and MOPC21 antibody was from ICN Pharmaceuticals (Paris, France).

    Techniques: Flow Cytometry, Cytometry, Expressing, Inhibition

    Engagement of CTLA-4 with antibody and stimulation of TCR by antigenic peptide upregulates TGF-β secretion. CD4 + T cell line CW-SW-1 specific for OVA peptide 323–339 was restimulated with OVA antigen (100 μg/ml) in the presence of normal BALB/c splenic APCs in X-Vivo-20. Anti–CTLA-4 (40 μg/ml) or the isotypic control hamster IgG ( Ham-IgG Ctrl ; 40 μg/ml) was added into the indicated culture wells. The supernatants were collected 72 h later and TGF-β was determined by ELISA.

    Journal: The Journal of Experimental Medicine

    Article Title: Engagement of Cytotoxic T Lymphocyte-associated Antigen 4 (CTLA-4) Induces Transforming Growth Factor ? (TGF-?) Production by Murine CD4+ T Cells

    doi:

    Figure Lengend Snippet: Engagement of CTLA-4 with antibody and stimulation of TCR by antigenic peptide upregulates TGF-β secretion. CD4 + T cell line CW-SW-1 specific for OVA peptide 323–339 was restimulated with OVA antigen (100 μg/ml) in the presence of normal BALB/c splenic APCs in X-Vivo-20. Anti–CTLA-4 (40 μg/ml) or the isotypic control hamster IgG ( Ham-IgG Ctrl ; 40 μg/ml) was added into the indicated culture wells. The supernatants were collected 72 h later and TGF-β was determined by ELISA.

    Article Snippet: For the staining of surface antigens, cells were incubated with FITC-conjugated anti–murine CD4 and PE-conjugated anti–CTLA-4 mAbs or their isotypic negative control antibodies as indicated on ice for 30 min. After washing twice with 1 ml of PBS-Az, cells were resuspended in 0.5 ml PBS-Az and analyzed on a FACScan® ( Becton Dickinson ).

    Techniques: Enzyme-linked Immunosorbent Assay

    Defect of CTLA-4–induced suppression of CD4 + T cell proliferation in TGF-β1 −/− mice. Freshly purified CD4 + T cells from asymptomatic TGF-β1 −/− mice were stimulated with the modified antibody regimen as indicated. Anti-CD3: 0.5 μg/ml; anti-CD28: 0.2 μg/ml; anti– CTLA-4 and hamster IgG ( Ham-IgG ): 20 μg/ml. Goat anti–hamster IgG (20 μg/ml) was added to all wells. ( A ) Surface expression of CTLA-4 on treated and control TGF-β1 −/− CD4 + T cells by flow cytometry. Cells were harvested after 56 h of culture and stained with FITC–anti-CD4 and PE–anti-CTLA-4. CD4 + T cells were gated and CTLA-4 staining of treated ( top ) and control ( middle ) cells on FL-2 channels is displayed. Bottom , negative control antibody staining for PE-conjugated anti-CTLA-4. ( B ) T cell proliferation of treated and control TGF-β1 −/− T cells. Data are expressed as mean ± SD ( CPM ) of triplicate wells.

    Journal: The Journal of Experimental Medicine

    Article Title: Engagement of Cytotoxic T Lymphocyte-associated Antigen 4 (CTLA-4) Induces Transforming Growth Factor ? (TGF-?) Production by Murine CD4+ T Cells

    doi:

    Figure Lengend Snippet: Defect of CTLA-4–induced suppression of CD4 + T cell proliferation in TGF-β1 −/− mice. Freshly purified CD4 + T cells from asymptomatic TGF-β1 −/− mice were stimulated with the modified antibody regimen as indicated. Anti-CD3: 0.5 μg/ml; anti-CD28: 0.2 μg/ml; anti– CTLA-4 and hamster IgG ( Ham-IgG ): 20 μg/ml. Goat anti–hamster IgG (20 μg/ml) was added to all wells. ( A ) Surface expression of CTLA-4 on treated and control TGF-β1 −/− CD4 + T cells by flow cytometry. Cells were harvested after 56 h of culture and stained with FITC–anti-CD4 and PE–anti-CTLA-4. CD4 + T cells were gated and CTLA-4 staining of treated ( top ) and control ( middle ) cells on FL-2 channels is displayed. Bottom , negative control antibody staining for PE-conjugated anti-CTLA-4. ( B ) T cell proliferation of treated and control TGF-β1 −/− T cells. Data are expressed as mean ± SD ( CPM ) of triplicate wells.

    Article Snippet: For the staining of surface antigens, cells were incubated with FITC-conjugated anti–murine CD4 and PE-conjugated anti–CTLA-4 mAbs or their isotypic negative control antibodies as indicated on ice for 30 min. After washing twice with 1 ml of PBS-Az, cells were resuspended in 0.5 ml PBS-Az and analyzed on a FACScan® ( Becton Dickinson ).

    Techniques: Mouse Assay, Purification, Modification, Expressing, Flow Cytometry, Cytometry, Staining, Negative Control

    CTLA-4 dose-dependent induction of TGF-β. ( A ) CD4 + Th2, Th1, and Th0 cell clones specific for OVA peptide 323–339 were restimulated with the antibody regimen as described for Fig. 3 A . TGF-β was determined by ELISA. The data are expressed as mean ± SD of duplicate cultures. ( B ) The CD4 + T cell clone 1A11 (Th0, Table 1 ) was stimulated with immobilized anti-CD3 (1 μg/ml) and different concentrations of anti–CTLA-4 or isotypic hamster IgG antibodies for 72 h. Supernatant TGF-β was determined in duplicate by ELISA, and the variations of the means are

    Journal: The Journal of Experimental Medicine

    Article Title: Engagement of Cytotoxic T Lymphocyte-associated Antigen 4 (CTLA-4) Induces Transforming Growth Factor ? (TGF-?) Production by Murine CD4+ T Cells

    doi:

    Figure Lengend Snippet: CTLA-4 dose-dependent induction of TGF-β. ( A ) CD4 + Th2, Th1, and Th0 cell clones specific for OVA peptide 323–339 were restimulated with the antibody regimen as described for Fig. 3 A . TGF-β was determined by ELISA. The data are expressed as mean ± SD of duplicate cultures. ( B ) The CD4 + T cell clone 1A11 (Th0, Table 1 ) was stimulated with immobilized anti-CD3 (1 μg/ml) and different concentrations of anti–CTLA-4 or isotypic hamster IgG antibodies for 72 h. Supernatant TGF-β was determined in duplicate by ELISA, and the variations of the means are

    Article Snippet: For the staining of surface antigens, cells were incubated with FITC-conjugated anti–murine CD4 and PE-conjugated anti–CTLA-4 mAbs or their isotypic negative control antibodies as indicated on ice for 30 min. After washing twice with 1 ml of PBS-Az, cells were resuspended in 0.5 ml PBS-Az and analyzed on a FACScan® ( Becton Dickinson ).

    Techniques: Clone Assay, Enzyme-linked Immunosorbent Assay

    TGF-β is associated with inhibition of CD4 +  T cell activation by CTLA-4 cross-linking. CD4 +  T cells isolated from B6 spleens  were cultured with anti-CD3 (2 μg/ml), anti-CD28 (5 μg/ml), and anti– CTLA-4 (20 μg/ml) followed by goat anti–hamster IgG ( Ham-IgG ; 20  μg/ml). Anti–TGF-β mAb or isotypic control antibody (mouse IgG1)  was included from the beginning of the culture at the indicated concentrations. Cells were cultured for 72 h. Data are expressed as mean cpm ±  SD of triplicate wells for [ 3 H]thymidine incorporation. Data not shown in  the figure are cpm of T cells incubated with medium alone (176 ± 26).  The experiments were repeated three times with similar results.

    Journal: The Journal of Experimental Medicine

    Article Title: Engagement of Cytotoxic T Lymphocyte-associated Antigen 4 (CTLA-4) Induces Transforming Growth Factor ? (TGF-?) Production by Murine CD4+ T Cells

    doi:

    Figure Lengend Snippet: TGF-β is associated with inhibition of CD4 + T cell activation by CTLA-4 cross-linking. CD4 + T cells isolated from B6 spleens were cultured with anti-CD3 (2 μg/ml), anti-CD28 (5 μg/ml), and anti– CTLA-4 (20 μg/ml) followed by goat anti–hamster IgG ( Ham-IgG ; 20 μg/ml). Anti–TGF-β mAb or isotypic control antibody (mouse IgG1) was included from the beginning of the culture at the indicated concentrations. Cells were cultured for 72 h. Data are expressed as mean cpm ± SD of triplicate wells for [ 3 H]thymidine incorporation. Data not shown in the figure are cpm of T cells incubated with medium alone (176 ± 26). The experiments were repeated three times with similar results.

    Article Snippet: For the staining of surface antigens, cells were incubated with FITC-conjugated anti–murine CD4 and PE-conjugated anti–CTLA-4 mAbs or their isotypic negative control antibodies as indicated on ice for 30 min. After washing twice with 1 ml of PBS-Az, cells were resuspended in 0.5 ml PBS-Az and analyzed on a FACScan® ( Becton Dickinson ).

    Techniques: Inhibition, Activation Assay, Isolation, Cell Culture, Incubation

    Cross-linking of CTLA-4 inhibits cytokine production by  CD4 +  T cells. CD4 +  T cells isolated from spleens of B6 mice were cultured  in complete DMEM only ( Med ) or with the indicated antibodies: anti– CTLA-4 (20 μg/ml) or control hamster IgG ( Ctrl ; 20 μg/ml) in the absence or presence of anti-CD3 (2 μg/ml) and anti-CD28 (5 μg/ml). Goat  anti–hamster IgG (heavy and light chains) antibody was then added to all  the wells at 20 μg/ml. T cell proliferation ( A ) was expressed as mean ± SD  of triplicate wells for  3 H incorporation ( CPM ). Secretion of IL-2 ( B ),  IFN-γ ( C ), and IL-4 ( D ) by CD4 +  T cells is shown. Supernatants were collected at 48 h, and the cytokine levels were determined by ELISA. The values are expressed as mean ± SD of replicate wells of ELISA plates.

    Journal: The Journal of Experimental Medicine

    Article Title: Engagement of Cytotoxic T Lymphocyte-associated Antigen 4 (CTLA-4) Induces Transforming Growth Factor ? (TGF-?) Production by Murine CD4+ T Cells

    doi:

    Figure Lengend Snippet: Cross-linking of CTLA-4 inhibits cytokine production by CD4 + T cells. CD4 + T cells isolated from spleens of B6 mice were cultured in complete DMEM only ( Med ) or with the indicated antibodies: anti– CTLA-4 (20 μg/ml) or control hamster IgG ( Ctrl ; 20 μg/ml) in the absence or presence of anti-CD3 (2 μg/ml) and anti-CD28 (5 μg/ml). Goat anti–hamster IgG (heavy and light chains) antibody was then added to all the wells at 20 μg/ml. T cell proliferation ( A ) was expressed as mean ± SD of triplicate wells for 3 H incorporation ( CPM ). Secretion of IL-2 ( B ), IFN-γ ( C ), and IL-4 ( D ) by CD4 + T cells is shown. Supernatants were collected at 48 h, and the cytokine levels were determined by ELISA. The values are expressed as mean ± SD of replicate wells of ELISA plates.

    Article Snippet: For the staining of surface antigens, cells were incubated with FITC-conjugated anti–murine CD4 and PE-conjugated anti–CTLA-4 mAbs or their isotypic negative control antibodies as indicated on ice for 30 min. After washing twice with 1 ml of PBS-Az, cells were resuspended in 0.5 ml PBS-Az and analyzed on a FACScan® ( Becton Dickinson ).

    Techniques: Isolation, Mouse Assay, Cell Culture, Enzyme-linked Immunosorbent Assay

    Egg deposition within PP disrupts lymphoid microarchitecture and causes loss of cellularity. (A) DsRed and eYFP expression demarcate B cell follicles and inter-follicular T cell zones of PP. Images are 3D rendered multi-photon confocal display of excised PP tissue derived from VaDsRed/CD19+ eYFP double fluorescent reporter mice (red = CD3+ cells; green = CD19+ cells; blue = second-harmonic generation by collagen fibres). Auto-fluorescent egg (arrows) proximal to PP at 6 and 8 weeks pi. All data are representative of two independent experiments, n = 3 mice. (B) Stereo fluorescent stereomicroscope images of PP in naïve, and infected CD3+/CD19+ reporter mice. Auto-fluorescent eggs (arrows) co-localise to T and B cell zones of PP at 14 weeks (lower panel). (C) 3D multiphoton confocal image of PP egg-granuloma (enlargement of section inset shown in B as dotted line); Sm = autofluorescent egg; arrows = cellular infiltrate; blue = second-harmonic generation by collagen fibres. (D) Numbers of CD11b+ cells, CD11b+SigLecF-F4/80+ macrophages, and CD11b+SigLecF+F4/80lo eosinophils in PP cell suspensions enumerated by flow cytometry in naïve mice, or after infection. (E) CD19+ PP follicle area in double reporter mice (min 3 PP/mouse). (F) Total viable PP, B220+ and IgA+B220+ B cell yields/mouse and (G) proportion of B220+ cells of total PP. (H) Stereo fluorescent stereomicroscope images of mLN in situ from naïve and infected CD3+ (red)/CD19+ (green) reporter mice. (I) mLN follicle area, (J) total viable cell and B220+ B cell yields in the mLN of naïve and infected CD3+T cell/CD19+B cell double reporter mice. All data; *P

    Journal: PLoS Pathogens

    Article Title: Blood Flukes Exploit Peyer's Patch Lymphoid Tissue to Facilitate Transmission from the Mammalian Host

    doi: 10.1371/journal.ppat.1003063

    Figure Lengend Snippet: Egg deposition within PP disrupts lymphoid microarchitecture and causes loss of cellularity. (A) DsRed and eYFP expression demarcate B cell follicles and inter-follicular T cell zones of PP. Images are 3D rendered multi-photon confocal display of excised PP tissue derived from VaDsRed/CD19+ eYFP double fluorescent reporter mice (red = CD3+ cells; green = CD19+ cells; blue = second-harmonic generation by collagen fibres). Auto-fluorescent egg (arrows) proximal to PP at 6 and 8 weeks pi. All data are representative of two independent experiments, n = 3 mice. (B) Stereo fluorescent stereomicroscope images of PP in naïve, and infected CD3+/CD19+ reporter mice. Auto-fluorescent eggs (arrows) co-localise to T and B cell zones of PP at 14 weeks (lower panel). (C) 3D multiphoton confocal image of PP egg-granuloma (enlargement of section inset shown in B as dotted line); Sm = autofluorescent egg; arrows = cellular infiltrate; blue = second-harmonic generation by collagen fibres. (D) Numbers of CD11b+ cells, CD11b+SigLecF-F4/80+ macrophages, and CD11b+SigLecF+F4/80lo eosinophils in PP cell suspensions enumerated by flow cytometry in naïve mice, or after infection. (E) CD19+ PP follicle area in double reporter mice (min 3 PP/mouse). (F) Total viable PP, B220+ and IgA+B220+ B cell yields/mouse and (G) proportion of B220+ cells of total PP. (H) Stereo fluorescent stereomicroscope images of mLN in situ from naïve and infected CD3+ (red)/CD19+ (green) reporter mice. (I) mLN follicle area, (J) total viable cell and B220+ B cell yields in the mLN of naïve and infected CD3+T cell/CD19+B cell double reporter mice. All data; *P

    Article Snippet: mLN cell recall assays Isolated mLN cells were seeded into 96-well plates (0.2×106 /well), in RPMI L-glutamine medium supplemented with 10% FCS, 50 µg/ml penicillin/streptomycin and stimulated with plate-bound anti-CD3 mAb (1 µg; Becton Dickinson), or SEA (50 µg/ml) as described (14).

    Techniques: Expressing, Derivative Assay, Mouse Assay, Infection, Flow Cytometry, Cytometry, In Situ

    PP are required to sustain optimum egg transmission and limit host morbidity. (A) Quantitative analysis of adult schistosome worm recoveries following perfusion of the hepatic portal system in WT or PP null mice. (B) Cytokine secretion by mLN cells from WT and PP null mice 8 weeks post-infection following in vitro stimulation with anti-CD3 mAb, or soluble egg antigen. (C) Masson's Trichrome stained histological sections of duodenum (blue = collagen) at post-infection in WT or PP null mice (red dotted lines = extent of egg granulomas). (D) Reduction in granuloma areas at 16 cf. 8 weeks; min 4 granulomas/mouse, **P

    Journal: PLoS Pathogens

    Article Title: Blood Flukes Exploit Peyer's Patch Lymphoid Tissue to Facilitate Transmission from the Mammalian Host

    doi: 10.1371/journal.ppat.1003063

    Figure Lengend Snippet: PP are required to sustain optimum egg transmission and limit host morbidity. (A) Quantitative analysis of adult schistosome worm recoveries following perfusion of the hepatic portal system in WT or PP null mice. (B) Cytokine secretion by mLN cells from WT and PP null mice 8 weeks post-infection following in vitro stimulation with anti-CD3 mAb, or soluble egg antigen. (C) Masson's Trichrome stained histological sections of duodenum (blue = collagen) at post-infection in WT or PP null mice (red dotted lines = extent of egg granulomas). (D) Reduction in granuloma areas at 16 cf. 8 weeks; min 4 granulomas/mouse, **P

    Article Snippet: mLN cell recall assays Isolated mLN cells were seeded into 96-well plates (0.2×106 /well), in RPMI L-glutamine medium supplemented with 10% FCS, 50 µg/ml penicillin/streptomycin and stimulated with plate-bound anti-CD3 mAb (1 µg; Becton Dickinson), or SEA (50 µg/ml) as described (14).

    Techniques: Transmission Assay, Mouse Assay, Infection, In Vitro, Staining

    Phenotype profiles of C-MSC and C-MSCγ. Cell surface marker expression levels were monitored by flow cytometry, as described in the “ Methods ” section. The open histogram with blue or green lines indicates CD105-, CD90-, CD73-, CD34-, CD45-, HLA-DR-, or CD86-positive cells. The IgG control is shown with a solid histogram. CD cluster of differentiation, C-MSC clumps of a mesenchymal stem cell/extracellular matrix complex cultured in growth medium for 3 days, C-MSCγ (50) C-MSC stimulated with 50 ng/mL IFN-γ for 24 h before the end of the culture period, HLA human leukocyte antigens

    Journal: Stem Cell Research & Therapy

    Article Title: Xenotransplantation of interferon-gamma-pretreated clumps of a human mesenchymal stem cell/extracellular matrix complex induces mouse calvarial bone regeneration

    doi: 10.1186/s13287-017-0550-1

    Figure Lengend Snippet: Phenotype profiles of C-MSC and C-MSCγ. Cell surface marker expression levels were monitored by flow cytometry, as described in the “ Methods ” section. The open histogram with blue or green lines indicates CD105-, CD90-, CD73-, CD34-, CD45-, HLA-DR-, or CD86-positive cells. The IgG control is shown with a solid histogram. CD cluster of differentiation, C-MSC clumps of a mesenchymal stem cell/extracellular matrix complex cultured in growth medium for 3 days, C-MSCγ (50) C-MSC stimulated with 50 ng/mL IFN-γ for 24 h before the end of the culture period, HLA human leukocyte antigens

    Article Snippet: The cells were then incubated with a mouse monoclonal anti-human CD90 IgG antibody (BD; 5E10), mouse monoclonal anti-human CD73 IgG antibody (BD; AD2), mouse monoclonal anti-human CD105 IgG antibody (Immunotools, Friesoythe, Germany; MEM-226), mouse monoclonal anti-CD34 IgG antibody (BD; 8G12), mouse monoclonal anti-CD45 IgG antibody (BD; 2D1), mouse anti-human human leukocyte antigen (HLA)-DR) IgG antibody (BD; G46-6), or mouse monoclonal anti-CD86 IgG antibody (BD; FUN-1) for 1 h at room temperature.

    Techniques: Marker, Expressing, Flow Cytometry, Cytometry, Cell Culture