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 "Transected Tendon Treated with a New Fibrin Sealant Alone or Associated with Adipose-Derived Stem Cells"

Article Title: Transected Tendon Treated with a New Fibrin Sealant Alone or Associated with Adipose-Derived Stem Cells

Journal: Cells

doi: 10.3390/cells8010056

In vitro differentiation potential of ASC ( n = 4) in 5P ( A ): adipogenic ( B ) and lipid stained with Sudan IV (→); chondrogenic ( C ) and proteoglycans stained with toluidine blue (▶); osteogenic ( D ) and calcium stained with alizarin red (▷). Different cells were stained after 4 weeks of culture. ( E ) Flow cytometry for ASC in 5P ( n = 4) with positive labeling for CD90 and CD105 and negative labeling for CD34. ( F ) Histograms demonstrate the x-axis fluorescence scale considered positive when the cell peak is above 101 (CD34) or 102 (CD90 and CD105). ( G ) Control for -APC, -PE and -FITC (with very low fluorescence), corresponding to non-marked cells. Bars = A, B, D: 120 μm; C: 40 μm.
Figure Legend Snippet: In vitro differentiation potential of ASC ( n = 4) in 5P ( A ): adipogenic ( B ) and lipid stained with Sudan IV (→); chondrogenic ( C ) and proteoglycans stained with toluidine blue (▶); osteogenic ( D ) and calcium stained with alizarin red (▷). Different cells were stained after 4 weeks of culture. ( E ) Flow cytometry for ASC in 5P ( n = 4) with positive labeling for CD90 and CD105 and negative labeling for CD34. ( F ) Histograms demonstrate the x-axis fluorescence scale considered positive when the cell peak is above 101 (CD34) or 102 (CD90 and CD105). ( G ) Control for -APC, -PE and -FITC (with very low fluorescence), corresponding to non-marked cells. Bars = A, B, D: 120 μm; C: 40 μm.

Techniques Used: In Vitro, Staining, Flow Cytometry, Cytometry, Labeling, Fluorescence

Immunofluorescence for CD90 and CD105 observed in the central portion of the TR of tendons on the 21st day ( n = 5). Groups N ( A – D ), T ( E – H ), FS ( I – L ), ASC ( M – P ) and FS + ASC ( Q – T ). Note CD90 and CD105 (→) positive marking in the transected region of ASC and FS + ASC groups. Bar = 50 μm.
Figure Legend Snippet: Immunofluorescence for CD90 and CD105 observed in the central portion of the TR of tendons on the 21st day ( n = 5). Groups N ( A – D ), T ( E – H ), FS ( I – L ), ASC ( M – P ) and FS + ASC ( Q – T ). Note CD90 and CD105 (→) positive marking in the transected region of ASC and FS + ASC groups. Bar = 50 μm.

Techniques Used: Immunofluorescence

4) Product Images from "Antigen-selective modulation of AAV immunogenicity with tolerogenic rapamycin nanoparticles enables successful vector re-administration"

Article Title: Antigen-selective modulation of AAV immunogenicity with tolerogenic rapamycin nanoparticles enables successful vector re-administration

Journal: Nature Communications

doi: 10.1038/s41467-018-06621-3

Inhibition of anti-AAV8 capsid cellular and humoral responses with SVP[Rapa] co-administration. a CD8 T cell infiltrates in the liver. Livers from animals treated in Fig. 1a were collected after killing on day 53 and evaluated for CD8 mRNA expression by quantitative PCR using the ΔΔC t method relative to housekeeping gene and to average of untreated mice. b – d Male C57BL/6 mice were treated with 4 × 10 12 vg kg − 1 of AAV8-VP1 vector together with SVP[Rapa] or with SVP[empty] control. 14 days later, spleens were collected for B and T cell assays. b Analysis of T cell recall responses after overnight stimulation with an AAV8 peptide pool in splenocytes measured by IFN-γ ELISpot and c anti-AAV8 IgG and IgM secreting B cell responses in splenocytes measured by B ELISpot. d Frequency of B220 + CD19 + B cells in spleens measured by flow cytometry. e – i Male C57BL/6 mice were treated with 4 × 10 12 vg kg − 1 of AAV8-luc vector together with SVP[Rapa] or with SVP[empty] control. 14 days later, animals were sacrificed. e Gating of germinal center (GC) B cells (CD95 + GL7 + ) are shown in representative flow cytometry plot. Cells were gated on B220 + IgD − cells. Shown is a mouse from the SVP[empty] control group. f Frequency of GC B cells (CD95 + GL7 + ) in lymph nodes of treated mice determined by flow cytometry as shown in e ; g frequency of CD25 + FoxP3 + regulatory T cells in lymph nodes; h frequency of CXCR5 + PD1 + Foxp3 + follicular regulatory T (Tfr) cells and i frequency of CXCR5 + PD1 + FoxP3 − follicular helper T (Tfh) cells in lymph nodes. SVP[Rapa] treatment consisted of 200 µg of rapamycin. Data are shown as mean ± s.d. Statistical analyses were performed by one-way ANOVA with Tukey post hoc test in a and by unpaired, two-tail t -test in b – d , f – i ( n = 5 in a – d , n = 20 in f – i . * p
Figure Legend Snippet: Inhibition of anti-AAV8 capsid cellular and humoral responses with SVP[Rapa] co-administration. a CD8 T cell infiltrates in the liver. Livers from animals treated in Fig. 1a were collected after killing on day 53 and evaluated for CD8 mRNA expression by quantitative PCR using the ΔΔC t method relative to housekeeping gene and to average of untreated mice. b – d Male C57BL/6 mice were treated with 4 × 10 12 vg kg − 1 of AAV8-VP1 vector together with SVP[Rapa] or with SVP[empty] control. 14 days later, spleens were collected for B and T cell assays. b Analysis of T cell recall responses after overnight stimulation with an AAV8 peptide pool in splenocytes measured by IFN-γ ELISpot and c anti-AAV8 IgG and IgM secreting B cell responses in splenocytes measured by B ELISpot. d Frequency of B220 + CD19 + B cells in spleens measured by flow cytometry. e – i Male C57BL/6 mice were treated with 4 × 10 12 vg kg − 1 of AAV8-luc vector together with SVP[Rapa] or with SVP[empty] control. 14 days later, animals were sacrificed. e Gating of germinal center (GC) B cells (CD95 + GL7 + ) are shown in representative flow cytometry plot. Cells were gated on B220 + IgD − cells. Shown is a mouse from the SVP[empty] control group. f Frequency of GC B cells (CD95 + GL7 + ) in lymph nodes of treated mice determined by flow cytometry as shown in e ; g frequency of CD25 + FoxP3 + regulatory T cells in lymph nodes; h frequency of CXCR5 + PD1 + Foxp3 + follicular regulatory T (Tfr) cells and i frequency of CXCR5 + PD1 + FoxP3 − follicular helper T (Tfh) cells in lymph nodes. SVP[Rapa] treatment consisted of 200 µg of rapamycin. Data are shown as mean ± s.d. Statistical analyses were performed by one-way ANOVA with Tukey post hoc test in a and by unpaired, two-tail t -test in b – d , f – i ( n = 5 in a – d , n = 20 in f – i . * p

Techniques Used: Inhibition, Expressing, Real-time Polymerase Chain Reaction, Mouse Assay, Plasmid Preparation, Enzyme-linked Immunospot, Flow Cytometry, Cytometry

5) Product Images from "Antigen-selective modulation of AAV immunogenicity with tolerogenic rapamycin nanoparticles enables successful vector re-administration"

Article Title: Antigen-selective modulation of AAV immunogenicity with tolerogenic rapamycin nanoparticles enables successful vector re-administration

Journal: Nature Communications

doi: 10.1038/s41467-018-06621-3

Inhibition of anti-AAV8 capsid cellular and humoral responses with SVP[Rapa] co-administration. a CD8 T cell infiltrates in the liver. Livers from animals treated in Fig. 1a were collected after killing on day 53 and evaluated for CD8 mRNA expression by quantitative PCR using the ΔΔC t method relative to housekeeping gene and to average of untreated mice. b – d Male C57BL/6 mice were treated with 4 × 10 12 vg kg − 1 of AAV8-VP1 vector together with SVP[Rapa] or with SVP[empty] control. 14 days later, spleens were collected for B and T cell assays. b Analysis of T cell recall responses after overnight stimulation with an AAV8 peptide pool in splenocytes measured by IFN-γ ELISpot and c anti-AAV8 IgG and IgM secreting B cell responses in splenocytes measured by B ELISpot. d Frequency of B220 + CD19 + B cells in spleens measured by flow cytometry. e – i Male C57BL/6 mice were treated with 4 × 10 12 vg kg − 1 of AAV8-luc vector together with SVP[Rapa] or with SVP[empty] control. 14 days later, animals were sacrificed. e Gating of germinal center (GC) B cells (CD95 + GL7 + ) are shown in representative flow cytometry plot. Cells were gated on B220 + IgD − cells. Shown is a mouse from the SVP[empty] control group. f Frequency of GC B cells (CD95 + GL7 + ) in lymph nodes of treated mice determined by flow cytometry as shown in e ; g frequency of CD25 + FoxP3 + regulatory T cells in lymph nodes; h frequency of CXCR5 + PD1 + Foxp3 + follicular regulatory T (Tfr) cells and i frequency of CXCR5 + PD1 + FoxP3 − follicular helper T (Tfh) cells in lymph nodes. SVP[Rapa] treatment consisted of 200 µg of rapamycin. Data are shown as mean ± s.d. Statistical analyses were performed by one-way ANOVA with Tukey post hoc test in a and by unpaired, two-tail t -test in b – d , f – i ( n = 5 in a – d , n = 20 in f – i . * p
Figure Legend Snippet: Inhibition of anti-AAV8 capsid cellular and humoral responses with SVP[Rapa] co-administration. a CD8 T cell infiltrates in the liver. Livers from animals treated in Fig. 1a were collected after killing on day 53 and evaluated for CD8 mRNA expression by quantitative PCR using the ΔΔC t method relative to housekeeping gene and to average of untreated mice. b – d Male C57BL/6 mice were treated with 4 × 10 12 vg kg − 1 of AAV8-VP1 vector together with SVP[Rapa] or with SVP[empty] control. 14 days later, spleens were collected for B and T cell assays. b Analysis of T cell recall responses after overnight stimulation with an AAV8 peptide pool in splenocytes measured by IFN-γ ELISpot and c anti-AAV8 IgG and IgM secreting B cell responses in splenocytes measured by B ELISpot. d Frequency of B220 + CD19 + B cells in spleens measured by flow cytometry. e – i Male C57BL/6 mice were treated with 4 × 10 12 vg kg − 1 of AAV8-luc vector together with SVP[Rapa] or with SVP[empty] control. 14 days later, animals were sacrificed. e Gating of germinal center (GC) B cells (CD95 + GL7 + ) are shown in representative flow cytometry plot. Cells were gated on B220 + IgD − cells. Shown is a mouse from the SVP[empty] control group. f Frequency of GC B cells (CD95 + GL7 + ) in lymph nodes of treated mice determined by flow cytometry as shown in e ; g frequency of CD25 + FoxP3 + regulatory T cells in lymph nodes; h frequency of CXCR5 + PD1 + Foxp3 + follicular regulatory T (Tfr) cells and i frequency of CXCR5 + PD1 + FoxP3 − follicular helper T (Tfh) cells in lymph nodes. SVP[Rapa] treatment consisted of 200 µg of rapamycin. Data are shown as mean ± s.d. Statistical analyses were performed by one-way ANOVA with Tukey post hoc test in a and by unpaired, two-tail t -test in b – d , f – i ( n = 5 in a – d , n = 20 in f – i . * p

Techniques Used: Inhibition, Expressing, Real-time Polymerase Chain Reaction, Mouse Assay, Plasmid Preparation, Enzyme-linked Immunospot, Flow Cytometry, Cytometry

6) Product Images from "Co-stimulatory function in primary germinal center responses: CD40 and B7 are required on distinct antigen-presenting cells"

Article Title: Co-stimulatory function in primary germinal center responses: CD40 and B7 are required on distinct antigen-presenting cells

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20161955

Antigen-specific T cell activation and Tfh cell development require CD40 on B cells but not DCs.  (A–D) Naive OT-II CD4 +  T cells were transferred to CD40 cKO mice followed by NP-OVA/Alum immunization 1 d later, and splenic OT-II cells were analyzed at day 8 after immunization. Recovered total OT-II cell number (A), FACS plot of OT-II (CD4 +  B220 −  CD45.1 +  Vα2 + ) cells analyzed for Tfh CXCR5 high  PD-1 high  phenotype (B), frequency of Tfh cells among OT-II (C), and total OT-II Tfh cell number were determined (D). Data are combined from three independent experiments. The total numbers of mice in the three combined experiments are CD40 flox ,  n  = 8; CD40 KO,  n  = 8; and DC-CD40 cKO,  n  = 11. ns, not significant. (E–H) A mixture of BM from B cell KO and CD40 KO donors was used to reconstitute irradiated B cell KO host mice, generating chimeras in which all B cells were constitutively CD40 deficient (CD40 −/−  + µMT). Control chimeras received a mixture of BM from B cell KO and WT donors (CD40 +/+  + µMT). OT-II T cells were transferred to recipient BM chimera mice (CD45.2) followed by NP-OVA/Alum immunization 1 d later, and splenic OT-II cells were analyzed at day 8 after immunization. Recovered OT-II cell number (E), FACS plot of OT-II (CD4 +  CD45.1 +  Vα2 + ) cells analyzed for the Tfh CXCR5 high PD-1 high  phenotype (F), frequency of OT-II Tfh cells among total CD4 +  T cells (G), and the number of Tfh phenotype OT-II cells were analyzed (H). Data presented are the combined result of three independent experiments. The total numbers of mice in the three combined experiments are unimmunized,  n  = 3; (CD40 +/+  + µMT) BM chimera,  n  = 3; and (CD40 −/−  + μMT) BM chimera,  n  = 5. Statistical significance was determined by Student’s  t  test. All error bars represent means ± SEM.
Figure Legend Snippet: Antigen-specific T cell activation and Tfh cell development require CD40 on B cells but not DCs. (A–D) Naive OT-II CD4 + T cells were transferred to CD40 cKO mice followed by NP-OVA/Alum immunization 1 d later, and splenic OT-II cells were analyzed at day 8 after immunization. Recovered total OT-II cell number (A), FACS plot of OT-II (CD4 + B220 − CD45.1 + Vα2 + ) cells analyzed for Tfh CXCR5 high PD-1 high phenotype (B), frequency of Tfh cells among OT-II (C), and total OT-II Tfh cell number were determined (D). Data are combined from three independent experiments. The total numbers of mice in the three combined experiments are CD40 flox , n = 8; CD40 KO, n = 8; and DC-CD40 cKO, n = 11. ns, not significant. (E–H) A mixture of BM from B cell KO and CD40 KO donors was used to reconstitute irradiated B cell KO host mice, generating chimeras in which all B cells were constitutively CD40 deficient (CD40 −/− + µMT). Control chimeras received a mixture of BM from B cell KO and WT donors (CD40 +/+ + µMT). OT-II T cells were transferred to recipient BM chimera mice (CD45.2) followed by NP-OVA/Alum immunization 1 d later, and splenic OT-II cells were analyzed at day 8 after immunization. Recovered OT-II cell number (E), FACS plot of OT-II (CD4 + CD45.1 + Vα2 + ) cells analyzed for the Tfh CXCR5 high PD-1 high phenotype (F), frequency of OT-II Tfh cells among total CD4 + T cells (G), and the number of Tfh phenotype OT-II cells were analyzed (H). Data presented are the combined result of three independent experiments. The total numbers of mice in the three combined experiments are unimmunized, n = 3; (CD40 +/+ + µMT) BM chimera, n = 3; and (CD40 −/− + μMT) BM chimera, n = 5. Statistical significance was determined by Student’s t test. All error bars represent means ± SEM.

Techniques Used: Activation Assay, Mouse Assay, FACS, Irradiation

CD40 on B cells but not DCs is required for TD antigen–specific IgG production.  (A) Generation of CD40 flox  mice. Exons 2 and 3 of the  CD40  gene on BAC were floxed by loxP sites (top). CD40 flox  BAC Tg mice were backcrossed to CD40 KO mice to eliminate endogenous CD40 expression. CD40 flox  BAC Tg expression on LPS-stimulated DCs and B cells is shown. (B) CD40 expression on LPS-stimulated splenic DCs and B cells of CD40 flox , CD40 flox  × CD11c-Cre Tg (DC-CD40 cKO), and CD40 KO mice. Splenocytes were stimulated with LPS for 48 h, and CD40 expression on B cells (B220 + ) and DCs (CD11c + ) was analyzed by flow cytometry. Filled histograms show anti-CD40 antibody staining of CD40 flox  or DC-CD40 cKO DCs and B cells. Dashed lines show anti-CD40 antibody staining of CD40 KO. (A and B) Data are representative of four independent experiments. (C) Antigen-specific IgG1 production of DC-CD40 cKO mice. Mice were immunized with NP-KLH/Alum, and serum was collected after 3 wk. Serum titer of anti-NP IgG1 was determined by ELISA. Dashed line indicates background OD value in empty wells. Data are combined results of four independent experiments. The total numbers of mice in the four combined experiments are CD40 flox ,  n  = 8; CD40 KO,  n  = 7; and DC-CD40 cKO,  n  = 6. ns, not significant. (D) Antibody affinity maturation was determined by the ratio of high-affinity NP-specific IgG1 to total NP-specific IgG1 in serum at 7 and 21 d after immunization. The total numbers of mice in the three combined experiments are CD40 flox ,  n  = 8; and DC-CD40 cKO,  n  = 6. (E) Mice were immunized with NP-KLH/Alum. At day 8 after immunization, the absolute number of NP-specific GC B cells (B220 +  GL7 +  CD38 dull  NP-PE + ) in the spleen was determined by flow cytometry. (D and E) Data presented are the combined result of three independent experiments. The total numbers of mice in the three combined experiments are CD40 flox ,  n  = 4; CD40 KO,  n  = 4; DC-CD40 cKO,  n  = 5; and unimmunized,  n  = 3. (F) Ex vivo IFN-γ production of purified splenic CD4 +  T cells after stimulation with PMA and ionomycin for 2 h. Representative FACS plots are shown. The graph is a combined result of three independent experiments. The total numbers of mice in the three combined experiments are  n  = 3 for each strain. (G) WT (CD45.1) + CD40 KO (CD45.2) mixed BM chimera mice were immunized with NP-KLH/Alum, and NP-specific GC B cells were analyzed 1 wk later. Each group,  n  = 4. Data are representative of two independent experiments. Statistical significance was determined by Student’s  t  test. All error bars represent means ± SEM.
Figure Legend Snippet: CD40 on B cells but not DCs is required for TD antigen–specific IgG production. (A) Generation of CD40 flox mice. Exons 2 and 3 of the CD40 gene on BAC were floxed by loxP sites (top). CD40 flox BAC Tg mice were backcrossed to CD40 KO mice to eliminate endogenous CD40 expression. CD40 flox BAC Tg expression on LPS-stimulated DCs and B cells is shown. (B) CD40 expression on LPS-stimulated splenic DCs and B cells of CD40 flox , CD40 flox × CD11c-Cre Tg (DC-CD40 cKO), and CD40 KO mice. Splenocytes were stimulated with LPS for 48 h, and CD40 expression on B cells (B220 + ) and DCs (CD11c + ) was analyzed by flow cytometry. Filled histograms show anti-CD40 antibody staining of CD40 flox or DC-CD40 cKO DCs and B cells. Dashed lines show anti-CD40 antibody staining of CD40 KO. (A and B) Data are representative of four independent experiments. (C) Antigen-specific IgG1 production of DC-CD40 cKO mice. Mice were immunized with NP-KLH/Alum, and serum was collected after 3 wk. Serum titer of anti-NP IgG1 was determined by ELISA. Dashed line indicates background OD value in empty wells. Data are combined results of four independent experiments. The total numbers of mice in the four combined experiments are CD40 flox , n = 8; CD40 KO, n = 7; and DC-CD40 cKO, n = 6. ns, not significant. (D) Antibody affinity maturation was determined by the ratio of high-affinity NP-specific IgG1 to total NP-specific IgG1 in serum at 7 and 21 d after immunization. The total numbers of mice in the three combined experiments are CD40 flox , n = 8; and DC-CD40 cKO, n = 6. (E) Mice were immunized with NP-KLH/Alum. At day 8 after immunization, the absolute number of NP-specific GC B cells (B220 + GL7 + CD38 dull NP-PE + ) in the spleen was determined by flow cytometry. (D and E) Data presented are the combined result of three independent experiments. The total numbers of mice in the three combined experiments are CD40 flox , n = 4; CD40 KO, n = 4; DC-CD40 cKO, n = 5; and unimmunized, n = 3. (F) Ex vivo IFN-γ production of purified splenic CD4 + T cells after stimulation with PMA and ionomycin for 2 h. Representative FACS plots are shown. The graph is a combined result of three independent experiments. The total numbers of mice in the three combined experiments are n = 3 for each strain. (G) WT (CD45.1) + CD40 KO (CD45.2) mixed BM chimera mice were immunized with NP-KLH/Alum, and NP-specific GC B cells were analyzed 1 wk later. Each group, n = 4. Data are representative of two independent experiments. Statistical significance was determined by Student’s t test. All error bars represent means ± SEM.

Techniques Used: Mouse Assay, BAC Assay, Expressing, Flow Cytometry, Cytometry, Staining, Enzyme-linked Immunosorbent Assay, Ex Vivo, Purification, FACS

7) Product Images from "Lenalidomide regulates CNS autoimmunity by promoting M2 macrophages polarization"

Article Title: Lenalidomide regulates CNS autoimmunity by promoting M2 macrophages polarization

Journal: Cell Death & Disease

doi: 10.1038/s41419-018-0290-x

The protective effect of lenalidomide on EAE is dependent on M2 phenotype. a WT mice were immunized with MOG 35–55 and treated with lenalidomide (30 mg/kg, i.g.) or vehicle (0.9% CMC-Na, i.g.) combined with empty or clodronate liposome (50 mg/kg, i.v.) at indicated time points (bottom arrows represent lenalidomide or vehicle treatment, upper arrows represent liposome treatment). Mean clinical score is shown ( n = 15 per group). b , c Flow cytometry analysis of M2 and M1 macrophages in spleen ( b ) and DLN ( c ) from vehicle- and lenalidomide-treated WT EAE mice at day 17. Representative FACS plots (left) and statistics from six mice per group (right) are shown; cells are gated for F4/80 + cells. d Flow cytometry analysis of M2 macrophages in whole spinal cord and brain. CNS-infiltrating MNCs isolated from vehicle- and lenalidomide-treated WT EAE mice at day 17 stained for antibodies, including CD11b, CD206, and CD45. CD11b + CD45 hi CD206 + cells were M2 macrophages. Representative FACS plots (left) and statistics from six mice per group (right) are shown. e WT mice were immunized with MOG 35–55 on day 0 and 3 × 10 6 BMDMs treated with or without 25 nM lenalidomide for 4 h were injected intravenously into these mice on day 9, 14, and 19 ( n = 5 per group). f The mean clinical score of mice in e . g BMDMs were treated with or without 25 nM lenalidomide for 4 h. Splenic CD4 + T cells were isolated from MOG-treated WT mice, labeled with1 μM CFSE and subsequently cocultured with BMDMs at a ratio of 1:4 supplemented with MOG 35–55 peptide (20 μg/ml) for 72 h. The proliferation of CD4 + T cells was confirmed by flow cytometry analysis based on CFSE dilution. Left panel represents flowcytometric dot plot of CFSE-labeled CD4 + T cells, right panel shows the percentage of CD4 + T cells that have proliferated based on CFSE dilution ( n = 3 per group). Data are presented as means ± SEM; * P
Figure Legend Snippet: The protective effect of lenalidomide on EAE is dependent on M2 phenotype. a WT mice were immunized with MOG 35–55 and treated with lenalidomide (30 mg/kg, i.g.) or vehicle (0.9% CMC-Na, i.g.) combined with empty or clodronate liposome (50 mg/kg, i.v.) at indicated time points (bottom arrows represent lenalidomide or vehicle treatment, upper arrows represent liposome treatment). Mean clinical score is shown ( n = 15 per group). b , c Flow cytometry analysis of M2 and M1 macrophages in spleen ( b ) and DLN ( c ) from vehicle- and lenalidomide-treated WT EAE mice at day 17. Representative FACS plots (left) and statistics from six mice per group (right) are shown; cells are gated for F4/80 + cells. d Flow cytometry analysis of M2 macrophages in whole spinal cord and brain. CNS-infiltrating MNCs isolated from vehicle- and lenalidomide-treated WT EAE mice at day 17 stained for antibodies, including CD11b, CD206, and CD45. CD11b + CD45 hi CD206 + cells were M2 macrophages. Representative FACS plots (left) and statistics from six mice per group (right) are shown. e WT mice were immunized with MOG 35–55 on day 0 and 3 × 10 6 BMDMs treated with or without 25 nM lenalidomide for 4 h were injected intravenously into these mice on day 9, 14, and 19 ( n = 5 per group). f The mean clinical score of mice in e . g BMDMs were treated with or without 25 nM lenalidomide for 4 h. Splenic CD4 + T cells were isolated from MOG-treated WT mice, labeled with1 μM CFSE and subsequently cocultured with BMDMs at a ratio of 1:4 supplemented with MOG 35–55 peptide (20 μg/ml) for 72 h. The proliferation of CD4 + T cells was confirmed by flow cytometry analysis based on CFSE dilution. Left panel represents flowcytometric dot plot of CFSE-labeled CD4 + T cells, right panel shows the percentage of CD4 + T cells that have proliferated based on CFSE dilution ( n = 3 per group). Data are presented as means ± SEM; * P

Techniques Used: Mouse Assay, Flow Cytometry, Cytometry, FACS, Isolation, Staining, Injection, Labeling

Lenalidomide suppresses autoimmune responses in EAE. a , b Flow cytometry analysis of Th1 and Th17 cells in spleen ( a ) and DLN ( b ) from vehicle- and lenalidomide-treated WT EAE mice at day 17. Representative fluorescence activated cell sorting (FACS) plots (left) and statistics from six mice per group (right) are shown; cells are gated for CD4 + T cells. c The total numbers of MNCs in whole spinal cord and brain were isolated from vehicle- and lenalidomide-treated mice on day 17 ( n = 6). d Flow cytometry analysis of Th1 and Th17 cells in CNS-infiltrating MNCs from vehicle- and lenalidomide-treated WT EAE mice at day 17. Representative FACS plots (left) and statistics from six mice per group (right) are shown; cells are gated for CD4 + T cells. Data are presented as means ± SEM; *P
Figure Legend Snippet: Lenalidomide suppresses autoimmune responses in EAE. a , b Flow cytometry analysis of Th1 and Th17 cells in spleen ( a ) and DLN ( b ) from vehicle- and lenalidomide-treated WT EAE mice at day 17. Representative fluorescence activated cell sorting (FACS) plots (left) and statistics from six mice per group (right) are shown; cells are gated for CD4 + T cells. c The total numbers of MNCs in whole spinal cord and brain were isolated from vehicle- and lenalidomide-treated mice on day 17 ( n = 6). d Flow cytometry analysis of Th1 and Th17 cells in CNS-infiltrating MNCs from vehicle- and lenalidomide-treated WT EAE mice at day 17. Representative FACS plots (left) and statistics from six mice per group (right) are shown; cells are gated for CD4 + T cells. Data are presented as means ± SEM; *P

Techniques Used: Flow Cytometry, Cytometry, Mouse Assay, Fluorescence, FACS, Isolation

8) Product Images from "Lack of FcRn Impairs Natural Killer Cell Development and Functions in the Tumor Microenvironment"

Article Title: Lack of FcRn Impairs Natural Killer Cell Development and Functions in the Tumor Microenvironment

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.02259

Influence of FcRn on NK cell functions and in vitro expansion. Purified splenic NK cells were analyzed by flow cytometry for (A) the intracellular measurement of IFN-γ and (B) the surface expression of the late endosomal marker CD107a, after 4-h incubation at 37°C without (Ctr) or with PMA (100 ng/mL)/ionomycine (500 ng/mL). Data are median ± Min to Max analyzed from eight independent experiments using pooled NK cells from 2 mice. Freshly isolated splenocytes were seeded in RMPI 1640 complete medium supplemented with 5,000 U/ml rhIL2 or with 5 ng/ml rhIL12, 50 ng/ml rhIL15, and 10 ng/ml rhIL18 for 4-h (C,D) . Within splenocytes, CD3 − /NK1.1 + /NKp46 + cells were analyzed for (C) the intracellular expression of IFN-γ and (D) the surface expression of CD107a by flow cytometry. Data are median ± Min to Max from two independent experiments using pooled spleens from 2 mice. (E) Cytotoxicity assay was performed against CFSE-labeled YAC-1 target cells with different ratios of purified NK cells previously maintained overnight in RPMI 1640 complete medium supplemented with 50 U/ml of rhIL2 ( n = 3). The results were expressed as means ± SEM. (F,G) Purified splenic NK cells were plated in complete medium supplemented with 5,000 U/ml rhIL2. (F) The living cell numbers and (G) the percentage of dead cells were determined daily by manual cell counting using trypan blue in Malassez chamber ( n = 3). The results were expressed as mean ± SEM (F) and median ± Min to Max (G) . ns = not significant * p ≤ 0.05 and ** p ≤ 0.005 using two-tailed non-parametric and unpaired Wilcoxon-Mann-Whitney test.
Figure Legend Snippet: Influence of FcRn on NK cell functions and in vitro expansion. Purified splenic NK cells were analyzed by flow cytometry for (A) the intracellular measurement of IFN-γ and (B) the surface expression of the late endosomal marker CD107a, after 4-h incubation at 37°C without (Ctr) or with PMA (100 ng/mL)/ionomycine (500 ng/mL). Data are median ± Min to Max analyzed from eight independent experiments using pooled NK cells from 2 mice. Freshly isolated splenocytes were seeded in RMPI 1640 complete medium supplemented with 5,000 U/ml rhIL2 or with 5 ng/ml rhIL12, 50 ng/ml rhIL15, and 10 ng/ml rhIL18 for 4-h (C,D) . Within splenocytes, CD3 − /NK1.1 + /NKp46 + cells were analyzed for (C) the intracellular expression of IFN-γ and (D) the surface expression of CD107a by flow cytometry. Data are median ± Min to Max from two independent experiments using pooled spleens from 2 mice. (E) Cytotoxicity assay was performed against CFSE-labeled YAC-1 target cells with different ratios of purified NK cells previously maintained overnight in RPMI 1640 complete medium supplemented with 50 U/ml of rhIL2 ( n = 3). The results were expressed as means ± SEM. (F,G) Purified splenic NK cells were plated in complete medium supplemented with 5,000 U/ml rhIL2. (F) The living cell numbers and (G) the percentage of dead cells were determined daily by manual cell counting using trypan blue in Malassez chamber ( n = 3). The results were expressed as mean ± SEM (F) and median ± Min to Max (G) . ns = not significant * p ≤ 0.05 and ** p ≤ 0.005 using two-tailed non-parametric and unpaired Wilcoxon-Mann-Whitney test.

Techniques Used: In Vitro, Purification, Flow Cytometry, Cytometry, Expressing, Marker, Incubation, Mouse Assay, Isolation, Cytotoxicity Assay, Labeling, Cell Counting, Two Tailed Test, MANN-WHITNEY

9) Product Images from "Fucoidan-Manganese Dioxide Nanoparticles Potentiate Radiation Therapy by Co-Targeting Tumor Hypoxia and Angiogenesis"

Article Title: Fucoidan-Manganese Dioxide Nanoparticles Potentiate Radiation Therapy by Co-Targeting Tumor Hypoxia and Angiogenesis

Journal: Marine Drugs

doi: 10.3390/md16120510

Effects of fucoidan-coated manganese dioxide nanoparticles (Fuco-MnO 2 -NPs) on ionizing radiation (IR) -induced apoptotic cell death in BxPC-3 cells. ( a ) Flow cytometry evaluation of cell cycle progression at 72 h post-irradiation. BxPC-3 cells were pretreated with 0.5 μg/mL of fucoidan and 1 μg/mL of PAH-MnO 2 -NPs and Fuco-MnO 2 -NPs for 3 h in a hypoxic (1% oxygen) or normoxic condition and then exposed to IR. 6 Gy of X-ray was applied because 4 Gy was not strong enough to induce apoptosis in BxPC-3 cells. After 72 h, cells were fixed and stained with PI. Histograms show the representative DNA content stained with PI. Stacked bar graphs show the relative percentage of cells at different cell cycle phases ( n = 3). ( b ) Apoptosis was assessed by percentage of Annexin-V-positive cells using flow cytometry. PAH-MnO 2 -NPs and Fuco-MnO 2 -NPs reversed suppression of IR-induced apoptosis by hypoxia ( n = 3). Data are presented as mean ± SD; * p
Figure Legend Snippet: Effects of fucoidan-coated manganese dioxide nanoparticles (Fuco-MnO 2 -NPs) on ionizing radiation (IR) -induced apoptotic cell death in BxPC-3 cells. ( a ) Flow cytometry evaluation of cell cycle progression at 72 h post-irradiation. BxPC-3 cells were pretreated with 0.5 μg/mL of fucoidan and 1 μg/mL of PAH-MnO 2 -NPs and Fuco-MnO 2 -NPs for 3 h in a hypoxic (1% oxygen) or normoxic condition and then exposed to IR. 6 Gy of X-ray was applied because 4 Gy was not strong enough to induce apoptosis in BxPC-3 cells. After 72 h, cells were fixed and stained with PI. Histograms show the representative DNA content stained with PI. Stacked bar graphs show the relative percentage of cells at different cell cycle phases ( n = 3). ( b ) Apoptosis was assessed by percentage of Annexin-V-positive cells using flow cytometry. PAH-MnO 2 -NPs and Fuco-MnO 2 -NPs reversed suppression of IR-induced apoptosis by hypoxia ( n = 3). Data are presented as mean ± SD; * p

Techniques Used: Flow Cytometry, Cytometry, Irradiation, Staining

10) Product Images from "Identification of Broad-Spectrum Antiviral Compounds by Targeting Viral Entry"

Article Title: Identification of Broad-Spectrum Antiviral Compounds by Targeting Viral Entry

Journal: Viruses

doi: 10.3390/v11020176

In vivo activity of Tyrphostin A9, Monensin, and Sofosbuvir. ( A ) Schematic of the drug treatment and infection regime in AG129 mice. Mice were treated with 5.7 mg/kg of Sofosbuvir, 10 mg/kg of Monensin, or 1mg/kg of Tyrphostin A9 before and after infection with 10 5 pfu of ZIKV, at the indicated intervals. Administration of Tyrphostin A9 was suspended at day 3 due to toxicity. Focus-forming units (ffu, top panels) and ZIKV RNA copies (bottom panels) per ml of serum at day 1, 3, and 7 p.i. upon treatment with Sofosbuvir ( B ), Monensin ( C ), or Tyrphostin A9 ( D ). N.D. (not detected), indicates that no infectious units were recovered. ZIKV RNA copies/ng at day 7 p.i. in the lymph nodes ( E ), liver ( F ), and brain ( G ) upon indicated treatment. Percentages of CD14 + CD11b + macrophages ( H ), CD14 + CD11c + DC ( I ), and CD3 + CD4 + T cells ( J ) infected with ZIKV at day 7 p.i. upon indicated treatment. P values from unpaired T tests are displayed for statistically significant comparisons.
Figure Legend Snippet: In vivo activity of Tyrphostin A9, Monensin, and Sofosbuvir. ( A ) Schematic of the drug treatment and infection regime in AG129 mice. Mice were treated with 5.7 mg/kg of Sofosbuvir, 10 mg/kg of Monensin, or 1mg/kg of Tyrphostin A9 before and after infection with 10 5 pfu of ZIKV, at the indicated intervals. Administration of Tyrphostin A9 was suspended at day 3 due to toxicity. Focus-forming units (ffu, top panels) and ZIKV RNA copies (bottom panels) per ml of serum at day 1, 3, and 7 p.i. upon treatment with Sofosbuvir ( B ), Monensin ( C ), or Tyrphostin A9 ( D ). N.D. (not detected), indicates that no infectious units were recovered. ZIKV RNA copies/ng at day 7 p.i. in the lymph nodes ( E ), liver ( F ), and brain ( G ) upon indicated treatment. Percentages of CD14 + CD11b + macrophages ( H ), CD14 + CD11c + DC ( I ), and CD3 + CD4 + T cells ( J ) infected with ZIKV at day 7 p.i. upon indicated treatment. P values from unpaired T tests are displayed for statistically significant comparisons.

Techniques Used: In Vivo, Activity Assay, Infection, Mouse Assay

11) Product Images from "Butyrate Attenuates Lung Inflammation by Negatively Modulating Th9 Cells"

Article Title: Butyrate Attenuates Lung Inflammation by Negatively Modulating Th9 Cells

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2019.00067

Butyrate treatment protects OVA-challenged mice from lung inflammation. Male C57BL/6 mice were intraperitoneally (IP) injected with OVA (30 μg) + Al(OH)3 (1.6 mg) at days 0 and 7. Mice also received 250 μl of either PBS or butyrate (1 M) (IP) at days 0, 1, 2, 7, 8, and 9. OVA-sensitized mice were nebulized with an OVA solution (3%) for 15 min at days 14, 15, and 16. The group that received butyrate during sensitization was also treated during challenge. A control group was sensitized and challenged without OVA. Euthanasia was performed 24 h after the last challenge. Lungs were digested and cells stained with monoclonal antibodies to determine the frequency of eosinophils (CD45+CD64-Ly6G-CD11b+Siglec-F+CD11c-) by flow cytometry (A) . Bar graph shows the frequency of eosinophils in the different groups (B) . Lung tissues were also stained with hematoxylin/eosin (H E) and periodic acid–Schiff (PAS), scale bars: 100 μm. Thick and thin arrows indicate inflammatory infiltrates and mucus production, respectively (C) . Data are shown as mean ± SD. One-way ANOVA followed by Tukey's multiple comparison test were used for statistical analysis. n = 5–7 mice per group.
Figure Legend Snippet: Butyrate treatment protects OVA-challenged mice from lung inflammation. Male C57BL/6 mice were intraperitoneally (IP) injected with OVA (30 μg) + Al(OH)3 (1.6 mg) at days 0 and 7. Mice also received 250 μl of either PBS or butyrate (1 M) (IP) at days 0, 1, 2, 7, 8, and 9. OVA-sensitized mice were nebulized with an OVA solution (3%) for 15 min at days 14, 15, and 16. The group that received butyrate during sensitization was also treated during challenge. A control group was sensitized and challenged without OVA. Euthanasia was performed 24 h after the last challenge. Lungs were digested and cells stained with monoclonal antibodies to determine the frequency of eosinophils (CD45+CD64-Ly6G-CD11b+Siglec-F+CD11c-) by flow cytometry (A) . Bar graph shows the frequency of eosinophils in the different groups (B) . Lung tissues were also stained with hematoxylin/eosin (H E) and periodic acid–Schiff (PAS), scale bars: 100 μm. Thick and thin arrows indicate inflammatory infiltrates and mucus production, respectively (C) . Data are shown as mean ± SD. One-way ANOVA followed by Tukey's multiple comparison test were used for statistical analysis. n = 5–7 mice per group.

Techniques Used: Mouse Assay, Injection, Staining, Flow Cytometry, Cytometry

12) Product Images from "Circadian Expression of Migratory Factors Establishes Lineage-Specific Signatures that Guide the Homing of Leukocyte Subsets to Tissues"

Article Title: Circadian Expression of Migratory Factors Establishes Lineage-Specific Signatures that Guide the Homing of Leukocyte Subsets to Tissues

Journal: Immunity

doi: 10.1016/j.immuni.2018.10.007

Relevance of Rhythmic Leukocyte Trafficking in Inflammation, Leukemia, and Humans (A) Blood leukocyte numbers after acute treatment without (ctrl) or with LPS in combination with functional blocking antibodies directed against the indicated molecules at ZT1 and ZT13 (n = 3–12 mice; one-way ANOVA followed by Dunnett comparison to the LPS group and unpaired Student’s t test for comparisons between ZT1 and ZT13 groups). (B) Overview of functional blocking effects on leukocyte subsets in blood after LPS treatment targeting the indicated molecules at ZT1 and ZT13 (n = 3–12 mice; one-way ANOVA). (C) Numbers of circulating blasts present in the blood of C57BL/6J CD45.1 recipients at midday 1 week after engraftment at ZT1 and ZT13 with mouse C1498 (AML) or BS50 (B-ALL) cells (n = 7 or 8 mice; Mann-Whitney test). (D) Numbers of circulating blasts present in the blood of NSG recipient mice at midday 1 week after engraftment at ZT1 and ZT13 with human NALM-6 B-ALL cells (n = 8 mice; unpaired Student’s t test). (E) Oscillation of blood B cell numbers in human blood (n = 8 subjects; repeated-measures one-way ANOVA). (F) CXCR4 expression on human B cells over 24 hr (n = 8 subjects; repeated-measures one-way ANOVA). (G) Transendothelial migration (TEM) capacity of human primary B cells harvested from three donors at 11 a.m. and 7 p.m. across HUVECs. Numbers are normalized to 11 a.m. levels (n = 4 assays; unpaired Student’s t test). (H and I) Blocking efficacy of AMD3100 (H) or an anti-LFA-1 antibody (I) on TEM capacity of human primary B cells harvested at 11 a.m. and 7 p.m. Numbers are normalized to and compared with those of vehicle and isotype controls, respectively (n = 3 donors; unpaired Student’s t test). (J and K) Example of the TEM capacity of human B cells from one patient at 11 a.m. and 7 p.m. after AMD3100 (J) or anti-LFA-1 treatment (K) plotted over time (n = 4 assays; two-way ANOVA with Tukey post-test). ∗ p
Figure Legend Snippet: Relevance of Rhythmic Leukocyte Trafficking in Inflammation, Leukemia, and Humans (A) Blood leukocyte numbers after acute treatment without (ctrl) or with LPS in combination with functional blocking antibodies directed against the indicated molecules at ZT1 and ZT13 (n = 3–12 mice; one-way ANOVA followed by Dunnett comparison to the LPS group and unpaired Student’s t test for comparisons between ZT1 and ZT13 groups). (B) Overview of functional blocking effects on leukocyte subsets in blood after LPS treatment targeting the indicated molecules at ZT1 and ZT13 (n = 3–12 mice; one-way ANOVA). (C) Numbers of circulating blasts present in the blood of C57BL/6J CD45.1 recipients at midday 1 week after engraftment at ZT1 and ZT13 with mouse C1498 (AML) or BS50 (B-ALL) cells (n = 7 or 8 mice; Mann-Whitney test). (D) Numbers of circulating blasts present in the blood of NSG recipient mice at midday 1 week after engraftment at ZT1 and ZT13 with human NALM-6 B-ALL cells (n = 8 mice; unpaired Student’s t test). (E) Oscillation of blood B cell numbers in human blood (n = 8 subjects; repeated-measures one-way ANOVA). (F) CXCR4 expression on human B cells over 24 hr (n = 8 subjects; repeated-measures one-way ANOVA). (G) Transendothelial migration (TEM) capacity of human primary B cells harvested from three donors at 11 a.m. and 7 p.m. across HUVECs. Numbers are normalized to 11 a.m. levels (n = 4 assays; unpaired Student’s t test). (H and I) Blocking efficacy of AMD3100 (H) or an anti-LFA-1 antibody (I) on TEM capacity of human primary B cells harvested at 11 a.m. and 7 p.m. Numbers are normalized to and compared with those of vehicle and isotype controls, respectively (n = 3 donors; unpaired Student’s t test). (J and K) Example of the TEM capacity of human B cells from one patient at 11 a.m. and 7 p.m. after AMD3100 (J) or anti-LFA-1 treatment (K) plotted over time (n = 4 assays; two-way ANOVA with Tukey post-test). ∗ p

Techniques Used: Functional Assay, Blocking Assay, Mouse Assay, MANN-WHITNEY, Expressing, Migration, Transmission Electron Microscopy

13) Product Images from "Antitumor Potential of Extracellular Vesicles Released by Genetically Modified Murine Colon Carcinoma Cells With Overexpression of Interleukin-12 and shRNA for TGF-β1"

Article Title: Antitumor Potential of Extracellular Vesicles Released by Genetically Modified Murine Colon Carcinoma Cells With Overexpression of Interleukin-12 and shRNA for TGF-β1

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2019.00211

The influence of immunotherapy on the immune landscape in MC38 tumor nodules. (A,F) Schemes of multiparameter flow cytometric analyses showing the way of distinguishing lymphoid (A) or myeloid (F) cell subpopulations. (B) The percentage of leukocytes in tumor nodules. (C–E,G,H,J) Percentages of effector or suppressor cell subpopulations which underwent changes during therapy. (I) The M1/M2 ratio showing changes in polarization of tumor-infiltrating macrophages occurring during therapy. To calculate the mean ± SD, 6–8 mice per group were analyzed. The differences between the groups were estimated using the nonparametric Kruskal-Wallis test followed by Dunn's multiple comparison test (* p
Figure Legend Snippet: The influence of immunotherapy on the immune landscape in MC38 tumor nodules. (A,F) Schemes of multiparameter flow cytometric analyses showing the way of distinguishing lymphoid (A) or myeloid (F) cell subpopulations. (B) The percentage of leukocytes in tumor nodules. (C–E,G,H,J) Percentages of effector or suppressor cell subpopulations which underwent changes during therapy. (I) The M1/M2 ratio showing changes in polarization of tumor-infiltrating macrophages occurring during therapy. To calculate the mean ± SD, 6–8 mice per group were analyzed. The differences between the groups were estimated using the nonparametric Kruskal-Wallis test followed by Dunn's multiple comparison test (* p

Techniques Used: Flow Cytometry, Mouse Assay

The method of isolation and characterization of TEx and TMv released by wild-type or genetically modified MC38. (A) Scheme of TEx and TMv isolation. (B) Representative density plots showing the method of evaluation and counting of CFSE stained TEx and TMv using the LSR Fortessa flow cytometer. The data are presented for the example of particles isolated from unmodified MC38 cells. TEM analysis of TEx (C,E) and TMv (D,F) counterstained with uranyl acetate (C,D) or with methylcellulose (E,F) . Magnification 100,000x. (G,H) Representative histograms showing the measurement of MC38-derived TEx and TMv particle size distribution using the DLS Zetasizer (Malvern). (I) WB analysis of CD81, CD9, TSG101, GM130, and calnexin in lysates from MC38 cell lines, TEx and TMv fractions. (J,K,N,O) Relative expression of mRNA for IL-12 or TGF-β1 in TEx and TMv isolated from wild-type or genetically modified MC38 cell lines. (L,M,P,Q) Concentration of IL-12 and TGF-β1 in lysates prepared from TEx and TMv isolated from wild-type or genetically modified MC38 cell lines measured using the ELISA. The results are given as the mean ± SD calculated for at least two repeats in two independent experiments. The differences between the groups were estimated using the nonparametric Kruskal-Wallis test followed by Dunn's multiple comparison test (* p
Figure Legend Snippet: The method of isolation and characterization of TEx and TMv released by wild-type or genetically modified MC38. (A) Scheme of TEx and TMv isolation. (B) Representative density plots showing the method of evaluation and counting of CFSE stained TEx and TMv using the LSR Fortessa flow cytometer. The data are presented for the example of particles isolated from unmodified MC38 cells. TEM analysis of TEx (C,E) and TMv (D,F) counterstained with uranyl acetate (C,D) or with methylcellulose (E,F) . Magnification 100,000x. (G,H) Representative histograms showing the measurement of MC38-derived TEx and TMv particle size distribution using the DLS Zetasizer (Malvern). (I) WB analysis of CD81, CD9, TSG101, GM130, and calnexin in lysates from MC38 cell lines, TEx and TMv fractions. (J,K,N,O) Relative expression of mRNA for IL-12 or TGF-β1 in TEx and TMv isolated from wild-type or genetically modified MC38 cell lines. (L,M,P,Q) Concentration of IL-12 and TGF-β1 in lysates prepared from TEx and TMv isolated from wild-type or genetically modified MC38 cell lines measured using the ELISA. The results are given as the mean ± SD calculated for at least two repeats in two independent experiments. The differences between the groups were estimated using the nonparametric Kruskal-Wallis test followed by Dunn's multiple comparison test (* p

Techniques Used: Isolation, Genetically Modified, Staining, Flow Cytometry, Cytometry, Transmission Electron Microscopy, Derivative Assay, Western Blot, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay

The influence of TEx or TMv isolated from wild-type MC38 cells or MC38 cells with overexpression of IL-12 and/or shTGFβ1 on dendritic cell (DC) activity in vitro . (A,B) Representative histograms and bar plots showing phenotypic changes of DCs after 24-h stimulation with TEx. (C–F) Splenocyte activity after primary stimulation with TEx-treated dendritic cells. The data show the percentage of CD8 + CD107a + CTLs (C) and CD49b + CD107a + NK cells (D) among splenocytes obtained after 5-day co-culture with TEx-treated DCs, their cytotoxic activity toward MC38 tumor cells (E) and production of IFN-γ during co-culture of splc and DCs (F) . (G,H) Representative histograms and bar plots showing phenotypic changes of DCs after 24-h stimulation with TMv. (I–L) Splenocyte activity after primary stimulation with TMv-treated dendritic cells. The data show the percentage of CD8 + CD107a + CTLs (I) and CD49b + CD107a + NK cells (J) among splenocytes obtained after 5-day co-culture with mTMv-treated DCs, their cytotoxic activity toward MC38 tumor cells (K) and production of IFN-γ during co-culture of splc and DCs (L) . The results are given as the mean ± SD calculated for three repeats in three independent experiments. The differences between the groups were estimated using the nonparametric Kruskal-Wallis test followed by Dunn's multiple comparison test (* p
Figure Legend Snippet: The influence of TEx or TMv isolated from wild-type MC38 cells or MC38 cells with overexpression of IL-12 and/or shTGFβ1 on dendritic cell (DC) activity in vitro . (A,B) Representative histograms and bar plots showing phenotypic changes of DCs after 24-h stimulation with TEx. (C–F) Splenocyte activity after primary stimulation with TEx-treated dendritic cells. The data show the percentage of CD8 + CD107a + CTLs (C) and CD49b + CD107a + NK cells (D) among splenocytes obtained after 5-day co-culture with TEx-treated DCs, their cytotoxic activity toward MC38 tumor cells (E) and production of IFN-γ during co-culture of splc and DCs (F) . (G,H) Representative histograms and bar plots showing phenotypic changes of DCs after 24-h stimulation with TMv. (I–L) Splenocyte activity after primary stimulation with TMv-treated dendritic cells. The data show the percentage of CD8 + CD107a + CTLs (I) and CD49b + CD107a + NK cells (J) among splenocytes obtained after 5-day co-culture with mTMv-treated DCs, their cytotoxic activity toward MC38 tumor cells (K) and production of IFN-γ during co-culture of splc and DCs (L) . The results are given as the mean ± SD calculated for three repeats in three independent experiments. The differences between the groups were estimated using the nonparametric Kruskal-Wallis test followed by Dunn's multiple comparison test (* p

Techniques Used: Isolation, Over Expression, Activity Assay, In Vitro, Co-Culture Assay

MC38 tumor growth after immunotherapy with mTEx and/or dendritic cells stimulated with mTMv. (A) Scheme of treatment. (B) Table presenting MC38 tumor growth inhibition (TGI) calculated on the 35th day of the therapeutic experiment in relation to the untreated group. (C) Curves presenting the mean tumor volume after immunotherapy. The differences between groups were estimated using the Friedman test (** p
Figure Legend Snippet: MC38 tumor growth after immunotherapy with mTEx and/or dendritic cells stimulated with mTMv. (A) Scheme of treatment. (B) Table presenting MC38 tumor growth inhibition (TGI) calculated on the 35th day of the therapeutic experiment in relation to the untreated group. (C) Curves presenting the mean tumor volume after immunotherapy. The differences between groups were estimated using the Friedman test (** p

Techniques Used: Inhibition

Effectiveness of IL-12 overexpression and TGF-β1 silencing in genetically modified MC38 cell lines. (A,C) Relative expression of IL-12 and TGF-β1 measured by real-time PCR in MC38 cells cultured in normoxic (A) or hypoxic (C) conditions. (B,D) Concentration of IL-12 and TGF-β1 in supernatants from MC38 cells cultured in normoxic (B) or hypoxic (D) conditions. The results are given as the mean ± SD calculated for at least two repeats in two independent experiments. The differences between the groups were estimated using the nonparametric Kruskal-Wallis test followed by Dunn's multiple comparison test (* p
Figure Legend Snippet: Effectiveness of IL-12 overexpression and TGF-β1 silencing in genetically modified MC38 cell lines. (A,C) Relative expression of IL-12 and TGF-β1 measured by real-time PCR in MC38 cells cultured in normoxic (A) or hypoxic (C) conditions. (B,D) Concentration of IL-12 and TGF-β1 in supernatants from MC38 cells cultured in normoxic (B) or hypoxic (D) conditions. The results are given as the mean ± SD calculated for at least two repeats in two independent experiments. The differences between the groups were estimated using the nonparametric Kruskal-Wallis test followed by Dunn's multiple comparison test (* p

Techniques Used: Over Expression, Genetically Modified, Expressing, Real-time Polymerase Chain Reaction, Cell Culture, Concentration Assay

14) Product Images from "Mucosal immunoglobulins protect the olfactory organ of teleost fish against parasitic infection"

Article Title: Mucosal immunoglobulins protect the olfactory organ of teleost fish against parasitic infection

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1007251

Kinetics of immune response and pathological changes in trout olfactory organ following Ich parasites infection. (A) Heat map illustrates results from quantitative real-time PCR of mRNAs for selected immune markers in parasite-challenged versus control fish measured at 1, 7, 14, 21, 28 and 75 days post infections with Ich parasite in the olfactory organ of rainbow trout ( n = 6 fish per group). Color value: log2 (fold change). (B) Relative expression of IgM, IgD and IgT at 1, 7, 28 and 75 days post infection with Ich parasite in olfactory organ of rainbow trout ( n = 6 fish per group). (C) Histological examination (haematoxylin eosin stain; H E) of the olfactory organ from ich-infected rainbow trout 7, 28, 75 d.p.i and uninfected fish ( n = 6 fish per group). Black arrows indicate the width of LP at the tip region (100 μm from the lamellar tip) of the olfactory lammella and red arrows indicate goblet cells. (D) The width of LP at the tip region the olfactory lamella in ich-infected rainbow trout 7, 28, 75 d.p.i and control fish counted from C. (E) The number of goblet cells at the olfactory lamella in ich-infected rainbow trout 7, 28, 75 d.p.i and control fish counted from C. * P
Figure Legend Snippet: Kinetics of immune response and pathological changes in trout olfactory organ following Ich parasites infection. (A) Heat map illustrates results from quantitative real-time PCR of mRNAs for selected immune markers in parasite-challenged versus control fish measured at 1, 7, 14, 21, 28 and 75 days post infections with Ich parasite in the olfactory organ of rainbow trout ( n = 6 fish per group). Color value: log2 (fold change). (B) Relative expression of IgM, IgD and IgT at 1, 7, 28 and 75 days post infection with Ich parasite in olfactory organ of rainbow trout ( n = 6 fish per group). (C) Histological examination (haematoxylin eosin stain; H E) of the olfactory organ from ich-infected rainbow trout 7, 28, 75 d.p.i and uninfected fish ( n = 6 fish per group). Black arrows indicate the width of LP at the tip region (100 μm from the lamellar tip) of the olfactory lammella and red arrows indicate goblet cells. (D) The width of LP at the tip region the olfactory lamella in ich-infected rainbow trout 7, 28, 75 d.p.i and control fish counted from C. (E) The number of goblet cells at the olfactory lamella in ich-infected rainbow trout 7, 28, 75 d.p.i and control fish counted from C. * P

Techniques Used: Infection, Real-time Polymerase Chain Reaction, Fluorescence In Situ Hybridization, Expressing, Staining

Immunoglobulin responses in the nasal mucus and serum from infected and survived trout. (A) Western blot analysis of IgT-, IgM- and IgD- specific binding to Ich in nasal mucus (dilution 1:2) from infected and survivor fish. (B and C) IgT-, IgM- and IgD- specific binding to Ich in dilutions of nasal mucus from infected (B) and survivor (C) fish, evaluated by densitometric analysis of immunoblots and presented as relative values to those of control fish ( n = 8 per group). (D) Western blot analysis of IgT-, IgM- and IgD- specific binding to Ich in serum (dilution 1:10) from infected and survivor fish. (E and F) IgT-, IgM- and IgD- specific binding to Ich in dilutions of serum from infected (E) and survivor (F) fish, evaluated by densitometric analysis of immunoblots and presented as relative values to those of control fish ( n = 8 per group). * P
Figure Legend Snippet: Immunoglobulin responses in the nasal mucus and serum from infected and survived trout. (A) Western blot analysis of IgT-, IgM- and IgD- specific binding to Ich in nasal mucus (dilution 1:2) from infected and survivor fish. (B and C) IgT-, IgM- and IgD- specific binding to Ich in dilutions of nasal mucus from infected (B) and survivor (C) fish, evaluated by densitometric analysis of immunoblots and presented as relative values to those of control fish ( n = 8 per group). (D) Western blot analysis of IgT-, IgM- and IgD- specific binding to Ich in serum (dilution 1:10) from infected and survivor fish. (E and F) IgT-, IgM- and IgD- specific binding to Ich in dilutions of serum from infected (E) and survivor (F) fish, evaluated by densitometric analysis of immunoblots and presented as relative values to those of control fish ( n = 8 per group). * P

Techniques Used: Infection, Western Blot, Binding Assay, Fluorescence In Situ Hybridization

Structural characterization of immunoglobulins in trout nasal mucus. (A) Fractionation of nasal mucus (~ 0.5 ml) by gel filtration (upper) followed by immunoblot analysis of the fractions with anti-trout IgM-, anti-trout IgD-specific mAbs, and anti-trout IgT-specific pAbs (lower). A 280 , absorbance at 280 nm. (B) SDS-PAGE of gel-filtration fractions (4–15%) corresponding to eluent (40 μl) at elution volumes of 8.5 ml and 11.5 ml under non-reducing conditions followed by immunoblot analysis with anti-trout IgM-, anti-trout IgD-specific mAbs or anti-trout IgT-specific pAbs. (C and D) Immunoblot and densitometric analysis of the concentration of IgT, IgM and IgD in nasal mucus (40 μl) (C) and serum (0.5 μl) (D) ( n = 12 fish). Ratio of IgT to IgM concentration (E) and IgD to IgM concentration (F) in nasal mucus and serum, calculated from the values shown in C and D. Results in figs C-F are expressed as mean and s.e.m. obtained from 12 individual fishes.
Figure Legend Snippet: Structural characterization of immunoglobulins in trout nasal mucus. (A) Fractionation of nasal mucus (~ 0.5 ml) by gel filtration (upper) followed by immunoblot analysis of the fractions with anti-trout IgM-, anti-trout IgD-specific mAbs, and anti-trout IgT-specific pAbs (lower). A 280 , absorbance at 280 nm. (B) SDS-PAGE of gel-filtration fractions (4–15%) corresponding to eluent (40 μl) at elution volumes of 8.5 ml and 11.5 ml under non-reducing conditions followed by immunoblot analysis with anti-trout IgM-, anti-trout IgD-specific mAbs or anti-trout IgT-specific pAbs. (C and D) Immunoblot and densitometric analysis of the concentration of IgT, IgM and IgD in nasal mucus (40 μl) (C) and serum (0.5 μl) (D) ( n = 12 fish). Ratio of IgT to IgM concentration (E) and IgD to IgM concentration (F) in nasal mucus and serum, calculated from the values shown in C and D. Results in figs C-F are expressed as mean and s.e.m. obtained from 12 individual fishes.

Techniques Used: Fractionation, Filtration, SDS Page, Concentration Assay, Fluorescence In Situ Hybridization

Local IgT-, IgM- and IgD-specific responses in olfactory organ explants of survivor fish. The olfactory organ, head kidney and spleen explants (~ 20 mg each) from control and survivor fish were cultured in medium (400 μl) for 7 days. Immunoblot analysis of IgT-, IgM- and IgD-specific binding to Ich in the culture medium of olfactory organ (A), head kidney (B) and spleen (C) (dilution 1:2) from control and survivor fish. (D-F) IgT-, IgM- and IgD-specific binding to Ich in dilutions of culture medium from olfactory organ (D), head kidney (E) and spleen (F) from control and survivor fish, measured by densitometric analysis of immunoblots and presented as relative values to those of control fish ( n = 6–8 per group). * P
Figure Legend Snippet: Local IgT-, IgM- and IgD-specific responses in olfactory organ explants of survivor fish. The olfactory organ, head kidney and spleen explants (~ 20 mg each) from control and survivor fish were cultured in medium (400 μl) for 7 days. Immunoblot analysis of IgT-, IgM- and IgD-specific binding to Ich in the culture medium of olfactory organ (A), head kidney (B) and spleen (C) (dilution 1:2) from control and survivor fish. (D-F) IgT-, IgM- and IgD-specific binding to Ich in dilutions of culture medium from olfactory organ (D), head kidney (E) and spleen (F) from control and survivor fish, measured by densitometric analysis of immunoblots and presented as relative values to those of control fish ( n = 6–8 per group). * P

Techniques Used: Fluorescence In Situ Hybridization, Cell Culture, Binding Assay, Western Blot

Increases of IgT + B cells and IgT concentration in the olfactory organ of trout infected with Ich. Percentage of IgT + and IgM + B cells in NALT (A) and head kidney (B) leukocytes of uninfected control fish, infected fish and survivor fish measured by flow cytometric analysis ( n = 12 per group). Concentration of IgT, IgM and IgD in nasal mucus (C) and serum (D) of control, infected and survivor fish ( n = 12 per group). * P
Figure Legend Snippet: Increases of IgT + B cells and IgT concentration in the olfactory organ of trout infected with Ich. Percentage of IgT + and IgM + B cells in NALT (A) and head kidney (B) leukocytes of uninfected control fish, infected fish and survivor fish measured by flow cytometric analysis ( n = 12 per group). Concentration of IgT, IgM and IgD in nasal mucus (C) and serum (D) of control, infected and survivor fish ( n = 12 per group). * P

Techniques Used: Concentration Assay, Infection, Fluorescence In Situ Hybridization, Flow Cytometry

IgT coats Ich parasite located in olfactory organ of infected trout. Four different microscope images (A-D) of slides immunofluorescence staining of Ich parasites in olfactory organ paraffinic sections from trout infected with Ich after 28 days ( n = 6). (A and B) Immunofluorescence stained with Ich (magenta), IgM (red) and IgT (green), nuclei stained with DAPI (blue) (from left to right). (C and D) Immunofluorescence stained with Ich (magenta), IgD (red) and IgT (green) with nuclei stained with DAPI (blue) (from left to right); DIC images showing merged staining (isotype-matched control antibody staining, S3A–S3C Fig ). Scale bars, 20 μm. Data are representative of at least three different independent experiments.
Figure Legend Snippet: IgT coats Ich parasite located in olfactory organ of infected trout. Four different microscope images (A-D) of slides immunofluorescence staining of Ich parasites in olfactory organ paraffinic sections from trout infected with Ich after 28 days ( n = 6). (A and B) Immunofluorescence stained with Ich (magenta), IgM (red) and IgT (green), nuclei stained with DAPI (blue) (from left to right). (C and D) Immunofluorescence stained with Ich (magenta), IgD (red) and IgT (green) with nuclei stained with DAPI (blue) (from left to right); DIC images showing merged staining (isotype-matched control antibody staining, S3A–S3C Fig ). Scale bars, 20 μm. Data are representative of at least three different independent experiments.

Techniques Used: Infection, Microscopy, Immunofluorescence, Staining

Accumulation of IgT + B cells in the olfactory organ of trout infected with Ich. DIC images of immunofluorescence staining on trout nasal paraffinic sections from uninfected fish (A), 28 days infected fish (B) and survivor fish (C), stained for IgT (green) and IgM (red); nuclei are stained with DAPI (blue). (D) Enlarged images of the areas outlined in c are showing some IgT + B cells possibly secreting IgT (white arrowhead) (isotype-matched control antibody staining, S1B Fig ). NC, nasal cavity; OE, olfactory epithelium; LP, lamina propria. Scale bar, 20 μm. Data are representative of at least three different independent experiments ( n = 8 per group).
Figure Legend Snippet: Accumulation of IgT + B cells in the olfactory organ of trout infected with Ich. DIC images of immunofluorescence staining on trout nasal paraffinic sections from uninfected fish (A), 28 days infected fish (B) and survivor fish (C), stained for IgT (green) and IgM (red); nuclei are stained with DAPI (blue). (D) Enlarged images of the areas outlined in c are showing some IgT + B cells possibly secreting IgT (white arrowhead) (isotype-matched control antibody staining, S1B Fig ). NC, nasal cavity; OE, olfactory epithelium; LP, lamina propria. Scale bar, 20 μm. Data are representative of at least three different independent experiments ( n = 8 per group).

Techniques Used: Infection, Immunofluorescence, Staining, Fluorescence In Situ Hybridization

Proliferative responses of IgT + and IgM + B cells in the olfactory organ of survived trout. Immunofluorescence analysis of EdU incorporation by IgT + or IgM + B cells in olfactory organ of control (A) and survivor fish (B). Nasal paraffin sections were stained for EdU (magenta), trout IgT (green), trout IgM (red) and nuclei (blue) detection ( n = 8 fish per group). NC, nasal cavity; OE, olfactory epithelium; LP, lamina propria. Scale bars, 20 μm. (C) Percentage of EdU + cells from total nasal cell in control or survivor fish counted from Fig 7A and 7B ( n = 8). (D) Percentage of EdU + cells from the total IgT + or IgM + B cells populations in olfactory organ of control and survivor fish counted from A and B. Data in A and B are representative of at least three independent experiments (mean and s.e.m.). Statistical analysis was performed by unpaired Student’s t -test. * P
Figure Legend Snippet: Proliferative responses of IgT + and IgM + B cells in the olfactory organ of survived trout. Immunofluorescence analysis of EdU incorporation by IgT + or IgM + B cells in olfactory organ of control (A) and survivor fish (B). Nasal paraffin sections were stained for EdU (magenta), trout IgT (green), trout IgM (red) and nuclei (blue) detection ( n = 8 fish per group). NC, nasal cavity; OE, olfactory epithelium; LP, lamina propria. Scale bars, 20 μm. (C) Percentage of EdU + cells from total nasal cell in control or survivor fish counted from Fig 7A and 7B ( n = 8). (D) Percentage of EdU + cells from the total IgT + or IgM + B cells populations in olfactory organ of control and survivor fish counted from A and B. Data in A and B are representative of at least three independent experiments (mean and s.e.m.). Statistical analysis was performed by unpaired Student’s t -test. * P

Techniques Used: Immunofluorescence, Fluorescence In Situ Hybridization, Staining

15) Product Images from "Deletion of Interleukin-4 Receptor Alpha-Responsive Keratinocytes in BALB/c Mice Does Not Alter Susceptibility to Cutaneous Leishmaniasis"

Article Title: Deletion of Interleukin-4 Receptor Alpha-Responsive Keratinocytes in BALB/c Mice Does Not Alter Susceptibility to Cutaneous Leishmaniasis

Journal: Infection and Immunity

doi: 10.1128/IAI.00710-18

Characterization of KRT14 cre IL-4Rα −/lox BALB/c mice. (A) Mouse breeding strategy. Transgenic BALB/c mice expressing cre-recombinase under the control of the KRT14 promoter were intercrossed with IL-4Rα −/− BALB/c mice and IL-4Rα lox/lox BALB/c mice to generate KRT14 cre IL-4Rα −/lox mice. (B) Genotyping by PCR analysis of tail DNA from KRT14 cre IL-4Rα −/lox , IL-4Rα −/lox , IL-4Rα +/+ , IL-4Rα −/− , and IL-4Rα lox/lox mice and a negative water control is shown. The yielded PCR products are indicated in base pairs. (C) Flow cytometry was performed to show IL-4Rα expression on ear keratinocytes isolated from naive mice. Keratinocytes were gated as CD45 − CD49 + K14 + . (D) Flow cytometry was performed to show IL-4Rα expression on nonkeratinocyte lymph node cells following L. major infection, staining for Th cells (CD3 + CD4 + ), B cells (CD19 + B220 + ), dendritic cells (CD11c + MHCII + ), and macrophages (CD11b + MHCII + ). (E) Dsc-1 mRNA expression in keratinocytes. Primary keratinocytes were isolated from tails of adult BALB/c, KRT14 cre IL-4Rα −/lox , and IL-4Rα −/− mice. Keratinocytes were left unstimulated (−) or stimulated (+) for 24 h with 20 ng/ml of recombinant IL-4. Cells were isolated and Dsc-1 mRNA expression was assessed via qRT-PCR. Values were normalized to hprt levels ( n = 3 in each group; representative of two individual experiments shown). Statistical analysis for the mRNA expression of Dsc-1 in keratinocytes was performed using a one-way analysis of variance (ANOVA), with Sidaks’s multiple-comparison test. **, P
Figure Legend Snippet: Characterization of KRT14 cre IL-4Rα −/lox BALB/c mice. (A) Mouse breeding strategy. Transgenic BALB/c mice expressing cre-recombinase under the control of the KRT14 promoter were intercrossed with IL-4Rα −/− BALB/c mice and IL-4Rα lox/lox BALB/c mice to generate KRT14 cre IL-4Rα −/lox mice. (B) Genotyping by PCR analysis of tail DNA from KRT14 cre IL-4Rα −/lox , IL-4Rα −/lox , IL-4Rα +/+ , IL-4Rα −/− , and IL-4Rα lox/lox mice and a negative water control is shown. The yielded PCR products are indicated in base pairs. (C) Flow cytometry was performed to show IL-4Rα expression on ear keratinocytes isolated from naive mice. Keratinocytes were gated as CD45 − CD49 + K14 + . (D) Flow cytometry was performed to show IL-4Rα expression on nonkeratinocyte lymph node cells following L. major infection, staining for Th cells (CD3 + CD4 + ), B cells (CD19 + B220 + ), dendritic cells (CD11c + MHCII + ), and macrophages (CD11b + MHCII + ). (E) Dsc-1 mRNA expression in keratinocytes. Primary keratinocytes were isolated from tails of adult BALB/c, KRT14 cre IL-4Rα −/lox , and IL-4Rα −/− mice. Keratinocytes were left unstimulated (−) or stimulated (+) for 24 h with 20 ng/ml of recombinant IL-4. Cells were isolated and Dsc-1 mRNA expression was assessed via qRT-PCR. Values were normalized to hprt levels ( n = 3 in each group; representative of two individual experiments shown). Statistical analysis for the mRNA expression of Dsc-1 in keratinocytes was performed using a one-way analysis of variance (ANOVA), with Sidaks’s multiple-comparison test. **, P

Techniques Used: Mouse Assay, Transgenic Assay, Expressing, Polymerase Chain Reaction, Flow Cytometry, Cytometry, Isolation, Infection, Staining, Recombinant, Quantitative RT-PCR

16) Product Images from "Differences of the Structure of Immune Regulatory Cell Populations between Cellular Material from Sonographically Detected Focal Thyroid Lesions and Peripheral Blood in Humans"

Article Title: Differences of the Structure of Immune Regulatory Cell Populations between Cellular Material from Sonographically Detected Focal Thyroid Lesions and Peripheral Blood in Humans

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms20040918

Exemplary plots of flow cytometry analysis showing the staining with antibodies specific for CD3 (T lymphocytes—red gates) and CD14 (monocytes—green gates) in mononuclear leukocyte fraction: ( A ) peripheral blood ( B ) FNAB.
Figure Legend Snippet: Exemplary plots of flow cytometry analysis showing the staining with antibodies specific for CD3 (T lymphocytes—red gates) and CD14 (monocytes—green gates) in mononuclear leukocyte fraction: ( A ) peripheral blood ( B ) FNAB.

Techniques Used: Flow Cytometry, Cytometry, Staining

Exemplary plots of flow cytometry analysis showing the staining with antibodies specific for CD14 and CD16 in monocyte fraction: ( A ) peripheral blood ( B ) FNAB. Gating of individual monocyte subpopulations is presented: classic monocytes (CD14 high CD16 − ), intermediate monocytes (CD14 high CD16 + ), non-classical monocytes (CD14 low CD16 + ).
Figure Legend Snippet: Exemplary plots of flow cytometry analysis showing the staining with antibodies specific for CD14 and CD16 in monocyte fraction: ( A ) peripheral blood ( B ) FNAB. Gating of individual monocyte subpopulations is presented: classic monocytes (CD14 high CD16 − ), intermediate monocytes (CD14 high CD16 + ), non-classical monocytes (CD14 low CD16 + ).

Techniques Used: Flow Cytometry, Cytometry, Staining

Percentage of CD14 + CD16 + monocytes in the leukocyte mononuclear fraction of peripheral blood and FNAB, respectively; in entire cohort and in particular patient groups; ( A ) all patients; ( B ) euthyroid patients (*** p
Figure Legend Snippet: Percentage of CD14 + CD16 + monocytes in the leukocyte mononuclear fraction of peripheral blood and FNAB, respectively; in entire cohort and in particular patient groups; ( A ) all patients; ( B ) euthyroid patients (*** p

Techniques Used:

17) Product Images from "IL22 furthers malignant transformation of rat mesenchymal stem cells, possibly in association with IL22RA1/STAT3 signaling"

Article Title: IL22 furthers malignant transformation of rat mesenchymal stem cells, possibly in association with IL22RA1/STAT3 signaling

Journal: Oncology Reports

doi: 10.3892/or.2019.7007

Phenotypic characterization of MSCs. Flow cytometric analyses of MSCs surface markers revealed that primary cells were positive for CD71 and CD90, while negative for CD34 and CD45. Control PE and Control FITC are the negative controls for PE and FITC staining, respectively. MSCs, mesenchymal stem cells; CD71, cluster of differentiation 71; FITC, fluorescein isothiocyanate; PE, phycoerythrin.
Figure Legend Snippet: Phenotypic characterization of MSCs. Flow cytometric analyses of MSCs surface markers revealed that primary cells were positive for CD71 and CD90, while negative for CD34 and CD45. Control PE and Control FITC are the negative controls for PE and FITC staining, respectively. MSCs, mesenchymal stem cells; CD71, cluster of differentiation 71; FITC, fluorescein isothiocyanate; PE, phycoerythrin.

Techniques Used: Flow Cytometry, Staining

18) Product Images from "Local Administration of GITR Agonistic Antibody Induces a Stronger Antitumor Immunity than Systemic Delivery"

Article Title: Local Administration of GITR Agonistic Antibody Induces a Stronger Antitumor Immunity than Systemic Delivery

Journal: Scientific Reports

doi: 10.1038/s41598-019-41724-x

Intratumoral injection of DTA-1 Ab increased T cell-infiltration into tumors. ( a ) Immnohistochemical staining of CT26 tumors. Seven days after the administration of DTA-1 Ab, the fresh frozen sections of subcutaneous tumors were processed for immunohistochemistry with anti-CD4 and anti-CD8 antibodies. ( b ) Number of CD4 + and CD8 + T cells in CT26 tumors. Positive cells were counted in 8 representative high power view fields (HPF, x 400) under microscope. P values for DTA-1 IP vs IT, as analysed by t -test. ( c ) Antitumor effect of intratumoral DTA-1 Ab injection after in vivo depletion of effector cells. Left panel: the depletion of CD4 + or CD8 + T cells (n = 7). Right panel: the depletion of NK cells (n = 7). P values for DTA-1 IT vs DTA-1 IT + anti-CD4 Ab (*), DTA-1 IT vs DTA-1 IT + anti-CD8 Ab (**) and DTA-1 IT vs DTA-1 IT + anti-asialoGM1 Ab (***), as analysed by t -test.
Figure Legend Snippet: Intratumoral injection of DTA-1 Ab increased T cell-infiltration into tumors. ( a ) Immnohistochemical staining of CT26 tumors. Seven days after the administration of DTA-1 Ab, the fresh frozen sections of subcutaneous tumors were processed for immunohistochemistry with anti-CD4 and anti-CD8 antibodies. ( b ) Number of CD4 + and CD8 + T cells in CT26 tumors. Positive cells were counted in 8 representative high power view fields (HPF, x 400) under microscope. P values for DTA-1 IP vs IT, as analysed by t -test. ( c ) Antitumor effect of intratumoral DTA-1 Ab injection after in vivo depletion of effector cells. Left panel: the depletion of CD4 + or CD8 + T cells (n = 7). Right panel: the depletion of NK cells (n = 7). P values for DTA-1 IT vs DTA-1 IT + anti-CD4 Ab (*), DTA-1 IT vs DTA-1 IT + anti-CD8 Ab (**) and DTA-1 IT vs DTA-1 IT + anti-asialoGM1 Ab (***), as analysed by t -test.

Techniques Used: Injection, Staining, Immunohistochemistry, Microscopy, In Vivo

Intratumoral injection of DTA-1 Ab more effectively activated tumor-specific immunity. ( a ) IFN-γ ELISpot assay in response to stimulation of CT26 tumor cells. DTA-1 Ab was injected into the CT26 subcutaneous tumor (IT) or into the peritoneal cavity (IP). Seven days later splenocytes were co-cultured with CT26 tumor cells, and stained with biotinylated anti-mouse IFN-γ antibody to detect captured IFN-γ (n = 3). P value for DTA-1 IP vs IT, as analysed by t -test. ( b ) The increase of tumor-specific CD8 + T cells in DTA-1 Ab IT-treated mice. The splenocytes were isolated seven days after DTA-1 Ab administration, and CT26-specific AH-1-tetramer-positive cells were analyzed by flow cytometry (upper panel). The ratio of AH-1 tetramer + cells among CD8 + cells (n = 4) (lower left panel). Total cell number of AH-1 tetramer + CD8 + cells in 1 × 10 4 events (n = 4) (lower middle panel). The ratio of p815 tetramer + cells among CD8 + cells (n = 4) (lower right panel). P values for PBS vs DTA-1 IP and DTA-1 IP vs IT, as analysed by t -test. ( c ) Re-challenge of CT26 tumor cells into the mice cured by the DTA-1 Ab treatment. CT26 cells were re-challenged at a few days after tumor rejection (n = 9).
Figure Legend Snippet: Intratumoral injection of DTA-1 Ab more effectively activated tumor-specific immunity. ( a ) IFN-γ ELISpot assay in response to stimulation of CT26 tumor cells. DTA-1 Ab was injected into the CT26 subcutaneous tumor (IT) or into the peritoneal cavity (IP). Seven days later splenocytes were co-cultured with CT26 tumor cells, and stained with biotinylated anti-mouse IFN-γ antibody to detect captured IFN-γ (n = 3). P value for DTA-1 IP vs IT, as analysed by t -test. ( b ) The increase of tumor-specific CD8 + T cells in DTA-1 Ab IT-treated mice. The splenocytes were isolated seven days after DTA-1 Ab administration, and CT26-specific AH-1-tetramer-positive cells were analyzed by flow cytometry (upper panel). The ratio of AH-1 tetramer + cells among CD8 + cells (n = 4) (lower left panel). Total cell number of AH-1 tetramer + CD8 + cells in 1 × 10 4 events (n = 4) (lower middle panel). The ratio of p815 tetramer + cells among CD8 + cells (n = 4) (lower right panel). P values for PBS vs DTA-1 IP and DTA-1 IP vs IT, as analysed by t -test. ( c ) Re-challenge of CT26 tumor cells into the mice cured by the DTA-1 Ab treatment. CT26 cells were re-challenged at a few days after tumor rejection (n = 9).

Techniques Used: Injection, Enzyme-linked Immunospot, Cell Culture, Staining, Mouse Assay, Isolation, Flow Cytometry, Cytometry

19) Product Images from "The effect of locally delivered cisplatin is dependent on an intact immune function in an experimental glioma model"

Article Title: The effect of locally delivered cisplatin is dependent on an intact immune function in an experimental glioma model

Journal: Scientific Reports

doi: 10.1038/s41598-019-42001-7

Cell viability and expression of MHC class I and II, CD80 and CD86 on GL261 cells following cisplatin exposure in vitro . GL261 cells were daily exposed to different doses of cisplatin for 3 days (72 hours). The following day, cells were analysed using flow cytometry for ( A ) viability using 7AAD staining (percentage of treated viable cells/non-treated viable cells, where the viability of the non-treated cells was regarded as 100%, mean values out of triplicates) or ( B ). No changes in expression of MHC class I, MHC class II, CD80 and CD86 following cisplatin exposure (1 μM), Histograms from 1 out of 3 experiments are presented.
Figure Legend Snippet: Cell viability and expression of MHC class I and II, CD80 and CD86 on GL261 cells following cisplatin exposure in vitro . GL261 cells were daily exposed to different doses of cisplatin for 3 days (72 hours). The following day, cells were analysed using flow cytometry for ( A ) viability using 7AAD staining (percentage of treated viable cells/non-treated viable cells, where the viability of the non-treated cells was regarded as 100%, mean values out of triplicates) or ( B ). No changes in expression of MHC class I, MHC class II, CD80 and CD86 following cisplatin exposure (1 μM), Histograms from 1 out of 3 experiments are presented.

Techniques Used: Expressing, In Vitro, Flow Cytometry, Cytometry, Staining

20) Product Images from "Effects of Pharmacological and Genetic Disruption of CXCR4 Chemokine Receptor Function in B-Cell Acute Lymphoblastic Leukaemia"

Article Title: Effects of Pharmacological and Genetic Disruption of CXCR4 Chemokine Receptor Function in B-Cell Acute Lymphoblastic Leukaemia

Journal: British journal of haematology

doi: 10.1111/bjh.14075

Bone Marrow Stromal Cell (BMSC) co-culture overcomes drug-induced cytotoxicity of B-ALL cell line and CXCR4 deletion reverses this effect (A) Dot plots show representative experiment depicting viability of dexamethasone (DEX) treated NALM6 cells in the presence and absence of BMSC co-culture. In the NALM6-BMSC co-culture sample, viability of NALM6 cells in supernatant and those that migrated beneath the stroma was measured separately. (B) Bar graphs show mean of three separate experiments illustrated in dot plots. In addition to DEX treatment, the experiment was also performed with 2.5 μM 4-HydroperoxyCyclophosphamide (4HC) and 2 nM vincristine (VIN) in the presence and absence (control) of BMSC co-culture. Bar diagrams represent mean drug induced cytotoxicity (± SEM) of three separate experiments. Viability was measured using PI/DiOC 6 staining at 48 h; asterisks indicate significant differences in cytotoxicity (* p
Figure Legend Snippet: Bone Marrow Stromal Cell (BMSC) co-culture overcomes drug-induced cytotoxicity of B-ALL cell line and CXCR4 deletion reverses this effect (A) Dot plots show representative experiment depicting viability of dexamethasone (DEX) treated NALM6 cells in the presence and absence of BMSC co-culture. In the NALM6-BMSC co-culture sample, viability of NALM6 cells in supernatant and those that migrated beneath the stroma was measured separately. (B) Bar graphs show mean of three separate experiments illustrated in dot plots. In addition to DEX treatment, the experiment was also performed with 2.5 μM 4-HydroperoxyCyclophosphamide (4HC) and 2 nM vincristine (VIN) in the presence and absence (control) of BMSC co-culture. Bar diagrams represent mean drug induced cytotoxicity (± SEM) of three separate experiments. Viability was measured using PI/DiOC 6 staining at 48 h; asterisks indicate significant differences in cytotoxicity (* p

Techniques Used: Co-Culture Assay, Staining

CXCR4 gene deletion significantly decreases chemotaxis and PEP of B-ALL cell lines (A) The mechanism of CXCR4 deletion through CRISPR-Cas9 gene editing system. The CRISPR Cas9 was directed towards the second extra cellular loop to introduce a frame-shift mutation resulting in lack of expression of the CXCR4 protein. (B) Histograms depict CXCR4 expression in NALM6 cells before and after CRISPR-Cas9 mediated CXCR4 knockout. Cells were stained with isotype control (black line) or CXCR4 antibody (shaded grey area). (C) NALM6 and TANOUE wild type (WT) and knockout (KO) cells (both untreated) were allowed to undergo chemotaxis towards 100 ng/ml CXCL12 or PEP beneath 9–15C Bone Marrow Stromal Cells and migrated cells were counted in flow cytometer for quantification. Bar diagrams representing mean chemotaxis/PEP (± SEM), with * p
Figure Legend Snippet: CXCR4 gene deletion significantly decreases chemotaxis and PEP of B-ALL cell lines (A) The mechanism of CXCR4 deletion through CRISPR-Cas9 gene editing system. The CRISPR Cas9 was directed towards the second extra cellular loop to introduce a frame-shift mutation resulting in lack of expression of the CXCR4 protein. (B) Histograms depict CXCR4 expression in NALM6 cells before and after CRISPR-Cas9 mediated CXCR4 knockout. Cells were stained with isotype control (black line) or CXCR4 antibody (shaded grey area). (C) NALM6 and TANOUE wild type (WT) and knockout (KO) cells (both untreated) were allowed to undergo chemotaxis towards 100 ng/ml CXCL12 or PEP beneath 9–15C Bone Marrow Stromal Cells and migrated cells were counted in flow cytometer for quantification. Bar diagrams representing mean chemotaxis/PEP (± SEM), with * p

Techniques Used: Chemotaxis Assay, CRISPR, Introduce, Mutagenesis, Expressing, Knock-Out, Staining, Flow Cytometry, Cytometry

CXCR4 inhibitors plerixafor (AMD3100) and BKT140 significantly reduce chemotaxis and Pseudoemperipolesis (PEP) of B-ALL cell lines and xenografts Representative phase contrast microscopy images (A) show SFO3 xenograft cells undergoing PEP in the presence or absence of CXCR4 inhibitor treatment (10 μg/ml). B-ALL cells were incubated in medium alone (control) or medium containing (10 μg/ml) plerixafor (AMD3100) or BKT140. The cells were allowed to (B) undergo chemotaxis towards 100 ng/ml CXCL12 or (A, C) undergo PEP beneath 9–15C Bone Marrow Stromal Cells and then counted in flow cytometer for quantification. The bar diagrams represents the mean chemotaxis/PEP (± SEM) of 6 B-ALL cell lines (left-hand graph) and 3 B-ALL xenografts (right-hand graph) in the presence or absence of CXCR4 inhibitors. Chemotaxis/PEP was significantly inhibited by both CXCR4 inhibitors (plerixafor/BKT140). (D) NALM6 (left panel) and RS4.11 (right panel) B-ALL cells undergoing PEP in the presence of plerixafor (P) or BKT140 (B) combined with CD49d antagonist CS-1, cells were incubated in medium alone (control), or medium supplemented with plerixafor (10 μg/ml), BKT140 (10 μg/ml), CS1 (10 μg/ml), or combinations of CS-1 with either CXCR4 antagonist, and then placed in BMSC co-cultures. Combination treatment with CXCR4 and CD49d antagonists inhibits B-ALL cell migration beneath BMSC (PEP) more effectively than single inhibitor treatment. The bar diagram represents the mean PEP (± SEM) (plerixafor/BKT140), with * p
Figure Legend Snippet: CXCR4 inhibitors plerixafor (AMD3100) and BKT140 significantly reduce chemotaxis and Pseudoemperipolesis (PEP) of B-ALL cell lines and xenografts Representative phase contrast microscopy images (A) show SFO3 xenograft cells undergoing PEP in the presence or absence of CXCR4 inhibitor treatment (10 μg/ml). B-ALL cells were incubated in medium alone (control) or medium containing (10 μg/ml) plerixafor (AMD3100) or BKT140. The cells were allowed to (B) undergo chemotaxis towards 100 ng/ml CXCL12 or (A, C) undergo PEP beneath 9–15C Bone Marrow Stromal Cells and then counted in flow cytometer for quantification. The bar diagrams represents the mean chemotaxis/PEP (± SEM) of 6 B-ALL cell lines (left-hand graph) and 3 B-ALL xenografts (right-hand graph) in the presence or absence of CXCR4 inhibitors. Chemotaxis/PEP was significantly inhibited by both CXCR4 inhibitors (plerixafor/BKT140). (D) NALM6 (left panel) and RS4.11 (right panel) B-ALL cells undergoing PEP in the presence of plerixafor (P) or BKT140 (B) combined with CD49d antagonist CS-1, cells were incubated in medium alone (control), or medium supplemented with plerixafor (10 μg/ml), BKT140 (10 μg/ml), CS1 (10 μg/ml), or combinations of CS-1 with either CXCR4 antagonist, and then placed in BMSC co-cultures. Combination treatment with CXCR4 and CD49d antagonists inhibits B-ALL cell migration beneath BMSC (PEP) more effectively than single inhibitor treatment. The bar diagram represents the mean PEP (± SEM) (plerixafor/BKT140), with * p

Techniques Used: Chemotaxis Assay, Microscopy, Incubation, Flow Cytometry, Cytometry, Migration

NALM6 CXCR4 Knockout cells display reduced leukaemia burden and increased survival (A) Nine mice per group were injected with GFP positive NALM6 CXCR4 wild type (WT) or NALM6 CXCR4 knockout (KO) cells and Bioluminescent Imaging (BLI) measured for all mice during the course of the experiment (on days 6, 10, 14 and 17). Right-hand graph depicts quantification of BLI (photons/second) for mice injected with NALM6 CXCR4 WT (blue line) vs. NALM6 CXCR4 KO (red line) (p
Figure Legend Snippet: NALM6 CXCR4 Knockout cells display reduced leukaemia burden and increased survival (A) Nine mice per group were injected with GFP positive NALM6 CXCR4 wild type (WT) or NALM6 CXCR4 knockout (KO) cells and Bioluminescent Imaging (BLI) measured for all mice during the course of the experiment (on days 6, 10, 14 and 17). Right-hand graph depicts quantification of BLI (photons/second) for mice injected with NALM6 CXCR4 WT (blue line) vs. NALM6 CXCR4 KO (red line) (p

Techniques Used: Knock-Out, Mouse Assay, Injection, Imaging

CXCR4 inhibitors sensitize NALM6 cells to chemotherapy (A) Bar graphs depict viability of NALM6 cells in supernatant, 48 h after treatment with 100 nM Dexamethasone (DEX), 2 nM Vincristine (VIN) and 2.5 μM 4-Hydroperoxy Cyclophosphamide (4HC) in the presence and absence (control) of bone marrow stromal cell (BMSC) co-culture. Additionally, before plating on stromal cells, NALM6 cells were incubated in medium alone or medium containing (10 μg/ml) plerixafor (AMD3100, P) or BKT140 (B). (B) Viability of NALM6 cells was analysed 48 h after treatment with 100 nM DEX or 2 nM VIN in the presence and absence (control) of CXCR4 ligand, CXCL12. Additionally, before the addition of CXCL12, NALM6 cells were incubated in medium alone or medium containing (10μg/ml) plerixafor (AMD3100) or BKT140. (C) NALM6 CXCR4 Wild type (WT) (black bars) and NALM6 CXCR4 Knockout (KO) (grey bars) were treated with 100 nM DEX (left hand side graph) or 2 nM VIN (right hand side graph) in the presence and absence (control) of BMSC co-culture and viability measured for cells in the supernatant. Bar diagrams represent mean drug induced cytotoxicity (± SEM) of three separate experiments. Viability was measured using PI/DiOC 6 staining at 48 h; asterisks indicate significant differences in cytotoxicity (* p
Figure Legend Snippet: CXCR4 inhibitors sensitize NALM6 cells to chemotherapy (A) Bar graphs depict viability of NALM6 cells in supernatant, 48 h after treatment with 100 nM Dexamethasone (DEX), 2 nM Vincristine (VIN) and 2.5 μM 4-Hydroperoxy Cyclophosphamide (4HC) in the presence and absence (control) of bone marrow stromal cell (BMSC) co-culture. Additionally, before plating on stromal cells, NALM6 cells were incubated in medium alone or medium containing (10 μg/ml) plerixafor (AMD3100, P) or BKT140 (B). (B) Viability of NALM6 cells was analysed 48 h after treatment with 100 nM DEX or 2 nM VIN in the presence and absence (control) of CXCR4 ligand, CXCL12. Additionally, before the addition of CXCL12, NALM6 cells were incubated in medium alone or medium containing (10μg/ml) plerixafor (AMD3100) or BKT140. (C) NALM6 CXCR4 Wild type (WT) (black bars) and NALM6 CXCR4 Knockout (KO) (grey bars) were treated with 100 nM DEX (left hand side graph) or 2 nM VIN (right hand side graph) in the presence and absence (control) of BMSC co-culture and viability measured for cells in the supernatant. Bar diagrams represent mean drug induced cytotoxicity (± SEM) of three separate experiments. Viability was measured using PI/DiOC 6 staining at 48 h; asterisks indicate significant differences in cytotoxicity (* p

Techniques Used: Co-Culture Assay, Incubation, Knock-Out, Staining

Expression of CXCR4 and CD49D in various ALL cell lines and xenograft cells Histograms show Mean Fluorescent Intensity Ratio (MFIR) of CXCR4 and CD49D (grey peaks) as compared to their respective isotype controls (black outlined peaks) in different B-ALL cell lines (A) and xenografts (B) . Mean Fluorescent Intensity Ratio (MFIR) is shown individually for each histogram.
Figure Legend Snippet: Expression of CXCR4 and CD49D in various ALL cell lines and xenograft cells Histograms show Mean Fluorescent Intensity Ratio (MFIR) of CXCR4 and CD49D (grey peaks) as compared to their respective isotype controls (black outlined peaks) in different B-ALL cell lines (A) and xenografts (B) . Mean Fluorescent Intensity Ratio (MFIR) is shown individually for each histogram.

Techniques Used: Expressing

21) Product Images from "Anti-TNF immunotherapy reduces CD8+ T cell-mediated antimicrobial activity against Mycobacterium tuberculosis in humans"

Article Title: Anti-TNF immunotherapy reduces CD8+ T cell-mediated antimicrobial activity against Mycobacterium tuberculosis in humans

Journal: The Journal of Clinical Investigation

doi: 10.1172/JCI38482

The majority of CD8 + and granulysin + or perforin + T cells are effector cells. PBMCs from healthy donors were stained with PerCP- or allophycocyanin-conjugated anti-CD8 and anti-granulysin (detected with donkey anti-rabbit biotin and streptavidin) or PE-conjugated perforin. Additional labeling was performed using allophycocyanin-conjugated anti-CD45RA and FITC-conjugated anti-CCR7 to detect T EM cells, PE-conjugated anti-CD161 and FITC-conjugated anti-Vα24 to detect NKT cells, and PE-conjugated CD56 and FITC-conjugated anti-CD16 to detect NK cells. Samples with appropriate isotypes were included in all experiments. ( A ) CD8 + granulysin + cells were gated, and the expression of additional markers within this gate was determined. Numbers denote the percentage of positive effector cells, NKT cells, or NK cells within the population of CD8 + granulysin + cells. For each sample, 1 × 10 6 cells were acquired. Shown is a typical result of 8 donors. ( B ) CD8 + perforin + cells were gated, and the expression of additional markers within this gate was determined. Numbers denote the percentage of positive effector cells, NKT cells, or NK cells within the population of CD8 + perforin + cells. For each sample, 1 × 10 6 cells were acquired. Shown is a typical result of 4 donors.
Figure Legend Snippet: The majority of CD8 + and granulysin + or perforin + T cells are effector cells. PBMCs from healthy donors were stained with PerCP- or allophycocyanin-conjugated anti-CD8 and anti-granulysin (detected with donkey anti-rabbit biotin and streptavidin) or PE-conjugated perforin. Additional labeling was performed using allophycocyanin-conjugated anti-CD45RA and FITC-conjugated anti-CCR7 to detect T EM cells, PE-conjugated anti-CD161 and FITC-conjugated anti-Vα24 to detect NKT cells, and PE-conjugated CD56 and FITC-conjugated anti-CD16 to detect NK cells. Samples with appropriate isotypes were included in all experiments. ( A ) CD8 + granulysin + cells were gated, and the expression of additional markers within this gate was determined. Numbers denote the percentage of positive effector cells, NKT cells, or NK cells within the population of CD8 + granulysin + cells. For each sample, 1 × 10 6 cells were acquired. Shown is a typical result of 8 donors. ( B ) CD8 + perforin + cells were gated, and the expression of additional markers within this gate was determined. Numbers denote the percentage of positive effector cells, NKT cells, or NK cells within the population of CD8 + perforin + cells. For each sample, 1 × 10 6 cells were acquired. Shown is a typical result of 4 donors.

Techniques Used: Staining, Labeling, Expressing

The majority of granulysin + cells in the peripheral blood are cytotoxic lymphocytes. PBMCs from healthy donors were stained with PE-Cy5-5–conjugated anti-CD4, PE-conjugated anti-CD56, allophycocyanin-conjugated anti-CD19, PerCP-conjugated anti-CD8 (filled histograms), or appropriate isotype controls (open histograms). All samples were then stained for granulysin using FITC-conjugated donkey anti-rabbit as a secondary antibody. Granulysin + cells were gated according to the isotype (control rabbit serum, not shown) and analyzed for the expression of cell surface markers. The percentages of CD4 + , CD56 + , CD19 + , and CD8 + cells within the granulysin gate are indicated. Shown is a typical result of 27 donors.
Figure Legend Snippet: The majority of granulysin + cells in the peripheral blood are cytotoxic lymphocytes. PBMCs from healthy donors were stained with PE-Cy5-5–conjugated anti-CD4, PE-conjugated anti-CD56, allophycocyanin-conjugated anti-CD19, PerCP-conjugated anti-CD8 (filled histograms), or appropriate isotype controls (open histograms). All samples were then stained for granulysin using FITC-conjugated donkey anti-rabbit as a secondary antibody. Granulysin + cells were gated according to the isotype (control rabbit serum, not shown) and analyzed for the expression of cell surface markers. The percentages of CD4 + , CD56 + , CD19 + , and CD8 + cells within the granulysin gate are indicated. Shown is a typical result of 27 donors.

Techniques Used: Staining, Expressing

Decreased frequency of T EMRA cells in patients treated with infliximab. PBMCs from patients with RA or AS were stained for CD8 (PE conjugated), CD45 (allophycocyanin conjugated), and CCR7 (FITC conjugated) before, 2 weeks, 3 months ( n = 7), and 1 year ( n = 5) after the beginning of infliximab therapy. ( A ) The percentage of all CD8 + T cells within the lymphocyte gate and the percentage of T EMRA cells within the CD8 gate were determined by flow cytometry. Shown is the percentage of CD8 + cells and T EMRA cells ( n = 7) of all donors tested. ( B ) Mean number of T EMRA cells within CD8 + T cells of the same patients before and during infliximab therapy. * P
Figure Legend Snippet: Decreased frequency of T EMRA cells in patients treated with infliximab. PBMCs from patients with RA or AS were stained for CD8 (PE conjugated), CD45 (allophycocyanin conjugated), and CCR7 (FITC conjugated) before, 2 weeks, 3 months ( n = 7), and 1 year ( n = 5) after the beginning of infliximab therapy. ( A ) The percentage of all CD8 + T cells within the lymphocyte gate and the percentage of T EMRA cells within the CD8 gate were determined by flow cytometry. Shown is the percentage of CD8 + cells and T EMRA cells ( n = 7) of all donors tested. ( B ) Mean number of T EMRA cells within CD8 + T cells of the same patients before and during infliximab therapy. * P

Techniques Used: Staining, Flow Cytometry, Cytometry

CD8 + T EMRA cells lyse M. tuberculosis –infected monocytes and reduce mycobacterial growth. CD8 + PBMCs were labeled with allophycocyanin-conjugated anti-CD45RA and FITC-conjugated anti-CCR7 and sorted into T EMRA , T EM , and T CM cell subpopulations. ( A ) Purified T cells were added as effector cells in a 51 Cr release assay using autologous M. tuberculosis –infected (MOI 5) or uninfected monocytes as target cells. Supernatants were harvested after 4 hours, and 51 Cr release was determined. Shown is mean ± SEM specific lysis of M. tuberculosis –infected monocytes of 4 independent experiments using different TST + donors. * P
Figure Legend Snippet: CD8 + T EMRA cells lyse M. tuberculosis –infected monocytes and reduce mycobacterial growth. CD8 + PBMCs were labeled with allophycocyanin-conjugated anti-CD45RA and FITC-conjugated anti-CCR7 and sorted into T EMRA , T EM , and T CM cell subpopulations. ( A ) Purified T cells were added as effector cells in a 51 Cr release assay using autologous M. tuberculosis –infected (MOI 5) or uninfected monocytes as target cells. Supernatants were harvested after 4 hours, and 51 Cr release was determined. Shown is mean ± SEM specific lysis of M. tuberculosis –infected monocytes of 4 independent experiments using different TST + donors. * P

Techniques Used: Infection, Labeling, Purification, Release Assay, Lysis

22) Product Images from "Anti-leukemia efficacy and mechanisms of action of SL-101, a novel anti-CD123 antibody-conjugate, in acute myeloid leukemia"

Article Title: Anti-leukemia efficacy and mechanisms of action of SL-101, a novel anti-CD123 antibody-conjugate, in acute myeloid leukemia

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

doi: 10.1158/1078-0432.CCR-16-1904

SL-101 efficiently kills primary AML blasts and stem/progenitor cells (A) Percentages of CD123 + and CD131 + fractions within CD45 dim blast gate on primary AML samples are shown. (B) Samples were treated with SL-101 at indicated doses for 48 hours. Normalized viable cell counts (left) were determined in a cohort of primary AML samples. (C) Gating scheme for the LSC population (CD45 dim CD34 + CD38 − CD123 + ). Specific apoptosis was calculated based on percentage of Annexin-V + cells using the following formula: percentage of specific apoptosis = 100 × (% apoptosis of treated cells − % apoptosis of control cells)/(100 − % apoptosis of control cells). Percentage of growth inhibition was calculated based on Annexin-V − /DAPI − viable cells using the following formula: 100 – 100 × % viable cells of treated cells/% of viable cells of control cells in both CD45 dim .
Figure Legend Snippet: SL-101 efficiently kills primary AML blasts and stem/progenitor cells (A) Percentages of CD123 + and CD131 + fractions within CD45 dim blast gate on primary AML samples are shown. (B) Samples were treated with SL-101 at indicated doses for 48 hours. Normalized viable cell counts (left) were determined in a cohort of primary AML samples. (C) Gating scheme for the LSC population (CD45 dim CD34 + CD38 − CD123 + ). Specific apoptosis was calculated based on percentage of Annexin-V + cells using the following formula: percentage of specific apoptosis = 100 × (% apoptosis of treated cells − % apoptosis of control cells)/(100 − % apoptosis of control cells). Percentage of growth inhibition was calculated based on Annexin-V − /DAPI − viable cells using the following formula: 100 – 100 × % viable cells of treated cells/% of viable cells of control cells in both CD45 dim .

Techniques Used: Inhibition

23) Product Images from "The calcineurin–NFAT pathway allows for urokinase receptor-mediated beta3 integrin signaling to cause podocyte injury"

Article Title: The calcineurin–NFAT pathway allows for urokinase receptor-mediated beta3 integrin signaling to cause podocyte injury

Journal: Journal of molecular medicine (Berlin, Germany)

doi: 10.1007/s00109-012-0960-6

NFATc1 affects β3 integrin activation, but not its surface expression. Ionomycin activation of NFAT was used as a positive control. Flow cytometry for ionomycin-treated podocytes showed that ionomycin (0.5–2 μM) activated β3 integrin (AP5 antibody labeling) in a dose-dependent manner ( a ) ( p
Figure Legend Snippet: NFATc1 affects β3 integrin activation, but not its surface expression. Ionomycin activation of NFAT was used as a positive control. Flow cytometry for ionomycin-treated podocytes showed that ionomycin (0.5–2 μM) activated β3 integrin (AP5 antibody labeling) in a dose-dependent manner ( a ) ( p

Techniques Used: Activation Assay, Expressing, Positive Control, Flow Cytometry, Cytometry, Antibody Labeling

CsA suppresses β3 integrin activation, but not its cell surface expression. Double immunofluorescence staining for active β3 integrin ( red ) and the podocyte marker synaptopodin ( synpo , green ) in glomeruli from NTX rats ( a ) and LPS mice ( b ]. AP5 labeling was strongly induced in podocytes form untreated NTX rats ( a ) or LPS mice ( b ). When treated with CsA, NTX rats ( a ) or LPS mice ( b ) showed a substantial reduction of AP5 labeling in podocytes. However, the total expression of β3 integrin ( red ) remained unchanged in glomeruli from NTX rats ( c ) and LPS mice ( d ) treated with or without CsA. ( e ) Flow cytometry for the AP5 antibody binding to cultured differentiated podocytes showed a high activated β3 integrin population after LPS treatment. CsA (0.25–1 μg/ml) reduced the activation of β3 integrin ( p
Figure Legend Snippet: CsA suppresses β3 integrin activation, but not its cell surface expression. Double immunofluorescence staining for active β3 integrin ( red ) and the podocyte marker synaptopodin ( synpo , green ) in glomeruli from NTX rats ( a ) and LPS mice ( b ]. AP5 labeling was strongly induced in podocytes form untreated NTX rats ( a ) or LPS mice ( b ). When treated with CsA, NTX rats ( a ) or LPS mice ( b ) showed a substantial reduction of AP5 labeling in podocytes. However, the total expression of β3 integrin ( red ) remained unchanged in glomeruli from NTX rats ( c ) and LPS mice ( d ) treated with or without CsA. ( e ) Flow cytometry for the AP5 antibody binding to cultured differentiated podocytes showed a high activated β3 integrin population after LPS treatment. CsA (0.25–1 μg/ml) reduced the activation of β3 integrin ( p

Techniques Used: Activation Assay, Expressing, Double Immunofluorescence Staining, Marker, Mouse Assay, Labeling, Flow Cytometry, Cytometry, Binding Assay, Cell Culture

NFAT inhibitor (11R-VIVIT) reduces proteinuria in LPS-induced proteinuric SCID mice. a Compared with untreated LPS mice, 11R-VIVIT-treated LPS mice showed a significant reduction in proteinuria in a dose-dependent manner with a maximum response at a dose of 15 mg kg −1 day −1 . b Double immunofluorescence staining for uPAR ( red ) and synaptopodin ( synpo , green ), a podocyte marker, in glomeruli from LPS mice. 11R-VIVIT inhibits uPAR expression. c Quantitative real-time RT-PCR performed on kidney glomerulus isolated from LPS mice shows that 11R-VIVIT inhibits Plaur mRNA (encoding uPAR) expression. d Double immunofluorescence staining for activeβ3 integrin (detected by AP5 antibody, red ) and synaptopodin ( synpo , green ) in glomeruli. 11R-VIVIT reduces AP5 labeling. e , f Neither 11R-VIVIT nor LPS fails to affect the expression of total β3 integrin protein ( red ) or ITGB3 mRNA (encoding β3 integrin). All values are expressed as the means±SD. * p
Figure Legend Snippet: NFAT inhibitor (11R-VIVIT) reduces proteinuria in LPS-induced proteinuric SCID mice. a Compared with untreated LPS mice, 11R-VIVIT-treated LPS mice showed a significant reduction in proteinuria in a dose-dependent manner with a maximum response at a dose of 15 mg kg −1 day −1 . b Double immunofluorescence staining for uPAR ( red ) and synaptopodin ( synpo , green ), a podocyte marker, in glomeruli from LPS mice. 11R-VIVIT inhibits uPAR expression. c Quantitative real-time RT-PCR performed on kidney glomerulus isolated from LPS mice shows that 11R-VIVIT inhibits Plaur mRNA (encoding uPAR) expression. d Double immunofluorescence staining for activeβ3 integrin (detected by AP5 antibody, red ) and synaptopodin ( synpo , green ) in glomeruli. 11R-VIVIT reduces AP5 labeling. e , f Neither 11R-VIVIT nor LPS fails to affect the expression of total β3 integrin protein ( red ) or ITGB3 mRNA (encoding β3 integrin). All values are expressed as the means±SD. * p

Techniques Used: Mouse Assay, Double Immunofluorescence Staining, Marker, Expressing, Quantitative RT-PCR, Isolation, Labeling

24) Product Images from "Involvement of the Dectin-1 Receptor upon the Effector Mechanisms of Human Phagocytic Cells against Paracoccidioides brasiliensis"

Article Title: Involvement of the Dectin-1 Receptor upon the Effector Mechanisms of Human Phagocytic Cells against Paracoccidioides brasiliensis

Journal: Journal of Immunology Research

doi: 10.1155/2019/1529189

Percentage of fungal recovery by colony-forming unit (CFU) analysis of neutrophil (PMN) cultures treated or not with IFN- γ (a), TNF- α (b), and GM-CSF (c) for 18 hours in the presence or absence of the anti-Dectin-1 monoclonal antibody (AD) and challenged with Pb265 for 4 hours. Data are expressed as the mean of 8 healthy volunteer donors tested. Statistical significance between groups is indicated.
Figure Legend Snippet: Percentage of fungal recovery by colony-forming unit (CFU) analysis of neutrophil (PMN) cultures treated or not with IFN- γ (a), TNF- α (b), and GM-CSF (c) for 18 hours in the presence or absence of the anti-Dectin-1 monoclonal antibody (AD) and challenged with Pb265 for 4 hours. Data are expressed as the mean of 8 healthy volunteer donors tested. Statistical significance between groups is indicated.

Techniques Used:

Hydrogen peroxide (H 2 O 2 ) production by monocytes (MO) treated or not with IFN- γ (a), TNF- α (b), and GM-CSF (c) for 18 hours in the presence or absence of the anti-Dectin-1 monoclonal antibody (AD) and challenged or not with Pb265 for 4 hours. Box-and-whisker plot showing data distribution of 8 healthy volunteer donors. Statistical significance between groups is indicated.
Figure Legend Snippet: Hydrogen peroxide (H 2 O 2 ) production by monocytes (MO) treated or not with IFN- γ (a), TNF- α (b), and GM-CSF (c) for 18 hours in the presence or absence of the anti-Dectin-1 monoclonal antibody (AD) and challenged or not with Pb265 for 4 hours. Box-and-whisker plot showing data distribution of 8 healthy volunteer donors. Statistical significance between groups is indicated.

Techniques Used: Whisker Assay

Histogram of a representative experiment for Dectin-1 receptor blockage for monocytes (a) and neutrophils (b), showing MFI of monocytes or neutrophil cultures (blue line) or monocytes or neutrophils treated with 3.0 μ g/mL (orange line) of anti-Dectin-1 monoclonal antibody. The red line represents MFI of the isotypic control.
Figure Legend Snippet: Histogram of a representative experiment for Dectin-1 receptor blockage for monocytes (a) and neutrophils (b), showing MFI of monocytes or neutrophil cultures (blue line) or monocytes or neutrophils treated with 3.0 μ g/mL (orange line) of anti-Dectin-1 monoclonal antibody. The red line represents MFI of the isotypic control.

Techniques Used:

Percentage of fungal recovery by colony-forming unit (CFU) analysis of monocyte (MO) cultures treated or not with IFN- γ (a), TNF- α (b), and GM-CSF (c) for 18 hours in the presence or absence of the anti-Dectin-1 monoclonal antibody (AD) and challenged with Pb265 for 4 hours. Data are expressed as the mean of 8 healthy volunteer donors tested. Statistical significance between groups is indicated.
Figure Legend Snippet: Percentage of fungal recovery by colony-forming unit (CFU) analysis of monocyte (MO) cultures treated or not with IFN- γ (a), TNF- α (b), and GM-CSF (c) for 18 hours in the presence or absence of the anti-Dectin-1 monoclonal antibody (AD) and challenged with Pb265 for 4 hours. Data are expressed as the mean of 8 healthy volunteer donors tested. Statistical significance between groups is indicated.

Techniques Used:

Dectin-1 receptor expression (MFI) of monocytes (MO) (a) and neutrophils (PMN) (b) treated or not with IFN- γ , TNF- α , and GM-CSF for 18 hours followed by the Pb265 challenge or not for 4 hours. Data are expressed as the mean of 8 healthy volunteer donors tested. ∗ Statistical significance between groups is indicated ( ∗ p
Figure Legend Snippet: Dectin-1 receptor expression (MFI) of monocytes (MO) (a) and neutrophils (PMN) (b) treated or not with IFN- γ , TNF- α , and GM-CSF for 18 hours followed by the Pb265 challenge or not for 4 hours. Data are expressed as the mean of 8 healthy volunteer donors tested. ∗ Statistical significance between groups is indicated ( ∗ p

Techniques Used: Expressing

Hydrogen peroxide (H 2 O 2 ) production by neutrophils (PMN) treated or not with IFN- γ (a), TNF- α (b), and GM-CSF (c) for 18 hours in the presence or absence of the anti-Dectin-1 monoclonal antibody (AD) and challenged or not with Pb265 for 4 hours. Box-and-whisker plot showing data distribution of 8 healthy volunteer donors. Statistical significance between groups is indicated.
Figure Legend Snippet: Hydrogen peroxide (H 2 O 2 ) production by neutrophils (PMN) treated or not with IFN- γ (a), TNF- α (b), and GM-CSF (c) for 18 hours in the presence or absence of the anti-Dectin-1 monoclonal antibody (AD) and challenged or not with Pb265 for 4 hours. Box-and-whisker plot showing data distribution of 8 healthy volunteer donors. Statistical significance between groups is indicated.

Techniques Used: Whisker Assay

25) Product Images from "Murine Lyme Arthritis Development Mediated by p38 Mitogen-Activated Protein Kinase Activity"

Article Title: Murine Lyme Arthritis Development Mediated by p38 Mitogen-Activated Protein Kinase Activity

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi:

B. burgdorferi -infected MKK3-deficient mice develop a lower inflammatory response. A , Two-week-infected mice were analyzed for IL-12 (filled bars), IFN- γ (shaded bars), and TNF- α (open bars) in the serum. B , The activation status of Mac-1 + and Ly6G + splenocytes was assessed by fluorescent staining of surface IFN- γ R α . Splenocytes were obtained from 2-wk-infected MKK3-deficient and wild-type controls with B. burgdorferi , double stained with anti-CD11b or anti-Ly6G plus anti-IFN- γ R α , and analyzed by flow cytometry. The results shown are representative of five to six mice in each group in two independent experiments.
Figure Legend Snippet: B. burgdorferi -infected MKK3-deficient mice develop a lower inflammatory response. A , Two-week-infected mice were analyzed for IL-12 (filled bars), IFN- γ (shaded bars), and TNF- α (open bars) in the serum. B , The activation status of Mac-1 + and Ly6G + splenocytes was assessed by fluorescent staining of surface IFN- γ R α . Splenocytes were obtained from 2-wk-infected MKK3-deficient and wild-type controls with B. burgdorferi , double stained with anti-CD11b or anti-Ly6G plus anti-IFN- γ R α , and analyzed by flow cytometry. The results shown are representative of five to six mice in each group in two independent experiments.

Techniques Used: Infection, Mouse Assay, Activation Assay, Staining, Flow Cytometry, Cytometry

26) Product Images from "Murine Lyme Arthritis Development Mediated by p38 Mitogen-Activated Protein Kinase Activity"

Article Title: Murine Lyme Arthritis Development Mediated by p38 Mitogen-Activated Protein Kinase Activity

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi:

A , IFN- γ and IL-4 production by CD4 + T cells in response to B. burgdorferi Ags in vitro. CD4 + T cells were purified from MKK3-deficient and wild-type controls at 2 wk of infection and restimulated in vitro with 10 μ g/ml of a B. burgdorferi lysate in the presence of syngeneic APCs. The supernatants were analyzed by capture ELISA for IFN- γ (filled bars) and IL-4 (open bars) at 40 h of stimulation. Results are the average of four mice and are representative of three independent experiments. B , B. burgdorferi -specific Ab isotype titers at 2 wk of infection in MKK3-deficient mice (open bars) and wild-type controls (filled bars). The results are representative of at least five experiments with similar results.
Figure Legend Snippet: A , IFN- γ and IL-4 production by CD4 + T cells in response to B. burgdorferi Ags in vitro. CD4 + T cells were purified from MKK3-deficient and wild-type controls at 2 wk of infection and restimulated in vitro with 10 μ g/ml of a B. burgdorferi lysate in the presence of syngeneic APCs. The supernatants were analyzed by capture ELISA for IFN- γ (filled bars) and IL-4 (open bars) at 40 h of stimulation. Results are the average of four mice and are representative of three independent experiments. B , B. burgdorferi -specific Ab isotype titers at 2 wk of infection in MKK3-deficient mice (open bars) and wild-type controls (filled bars). The results are representative of at least five experiments with similar results.

Techniques Used: In Vitro, Purification, Infection, Enzyme-linked Immunosorbent Assay, Mouse Assay

B. burgdorferi -infected MKK3-deficient mice develop a lower inflammatory response. A , Two-week-infected mice were analyzed for IL-12 (filled bars), IFN- γ (shaded bars), and TNF- α (open bars) in the serum. B , The activation status of Mac-1 + and Ly6G + splenocytes was assessed by fluorescent staining of surface IFN- γ R α . Splenocytes were obtained from 2-wk-infected MKK3-deficient and wild-type controls with B. burgdorferi , double stained with anti-CD11b or anti-Ly6G plus anti-IFN- γ R α , and analyzed by flow cytometry. The results shown are representative of five to six mice in each group in two independent experiments.
Figure Legend Snippet: B. burgdorferi -infected MKK3-deficient mice develop a lower inflammatory response. A , Two-week-infected mice were analyzed for IL-12 (filled bars), IFN- γ (shaded bars), and TNF- α (open bars) in the serum. B , The activation status of Mac-1 + and Ly6G + splenocytes was assessed by fluorescent staining of surface IFN- γ R α . Splenocytes were obtained from 2-wk-infected MKK3-deficient and wild-type controls with B. burgdorferi , double stained with anti-CD11b or anti-Ly6G plus anti-IFN- γ R α , and analyzed by flow cytometry. The results shown are representative of five to six mice in each group in two independent experiments.

Techniques Used: Infection, Mouse Assay, Activation Assay, Staining, Flow Cytometry, Cytometry

27) Product Images from "Smap1 deficiency perturbs receptor trafficking and predisposes mice to myelodysplasia"

Article Title: Smap1 deficiency perturbs receptor trafficking and predisposes mice to myelodysplasia

Journal: The Journal of Clinical Investigation

doi: 10.1172/JCI63711

Transport kinetics and c-KIT signaling in BMMCs. ( A ) Endocytosis of c-KIT. Smap1 +/+ and Smap1 –/– BMMCs were cultured, starved in the presence of cycloheximide, incubated with SCF at 37°C for the indicated times, and processed for flow cytometry analyses. The top panel displays the fluorescence intensity of c-KIT and cell numbers, whereas the bottom panel plots the percentages of internalized c-KIT calculated by considering the initial surface fluorescence to be 100%. BMMCs were prepared from 3 independent pairs of Smap1 +/+ and Smap1 –/– mice and processed for assays. Averages ± SD of internalized c-KIT were calculated for each incubation time ( n = 3). ( B ) Immunofluorescence detection of c-KIT in BMMCs. The Smap1 +/+ and Smap1 –/– cells were incubated in the presence of SCF for the indicated times and stained for c-KIT. Scale bar: 10 μm. ( C ) Activation status of c-KIT signaling molecules. Wild-type and Smap1 –/– BMMCs were incubated with SCF for the indicated times, and protein lysates were prepared and processed for immunoprecipitation/immunoblot analyses. Band densities were quantified, and averages ± SD are shown ( n = 3). p-c-KIT, phosphorylated form of c-KIT; p-ERK1/2, phosphorylated form of ERK1/2; ub-c-KIT, ubiquitinylated c-KIT; c-KIT-associated Grb2, Grb2 recruited into anti–c-KIT immunoprecipitates. ( D ) DNA synthesis in BMMCs. Triplicate cultures of cells were prepared from each of the wild-type and Smap1 –/– mice, incubated in the presence of IL-3 and/or SCF for 16 hours, and then treated with 3 H-thymidine for 8 hours. The incorporation of 3 H-thymidine into acid-insoluble fractions was measured, and averages ± SD are shown ( n = 3). * P
Figure Legend Snippet: Transport kinetics and c-KIT signaling in BMMCs. ( A ) Endocytosis of c-KIT. Smap1 +/+ and Smap1 –/– BMMCs were cultured, starved in the presence of cycloheximide, incubated with SCF at 37°C for the indicated times, and processed for flow cytometry analyses. The top panel displays the fluorescence intensity of c-KIT and cell numbers, whereas the bottom panel plots the percentages of internalized c-KIT calculated by considering the initial surface fluorescence to be 100%. BMMCs were prepared from 3 independent pairs of Smap1 +/+ and Smap1 –/– mice and processed for assays. Averages ± SD of internalized c-KIT were calculated for each incubation time ( n = 3). ( B ) Immunofluorescence detection of c-KIT in BMMCs. The Smap1 +/+ and Smap1 –/– cells were incubated in the presence of SCF for the indicated times and stained for c-KIT. Scale bar: 10 μm. ( C ) Activation status of c-KIT signaling molecules. Wild-type and Smap1 –/– BMMCs were incubated with SCF for the indicated times, and protein lysates were prepared and processed for immunoprecipitation/immunoblot analyses. Band densities were quantified, and averages ± SD are shown ( n = 3). p-c-KIT, phosphorylated form of c-KIT; p-ERK1/2, phosphorylated form of ERK1/2; ub-c-KIT, ubiquitinylated c-KIT; c-KIT-associated Grb2, Grb2 recruited into anti–c-KIT immunoprecipitates. ( D ) DNA synthesis in BMMCs. Triplicate cultures of cells were prepared from each of the wild-type and Smap1 –/– mice, incubated in the presence of IL-3 and/or SCF for 16 hours, and then treated with 3 H-thymidine for 8 hours. The incorporation of 3 H-thymidine into acid-insoluble fractions was measured, and averages ± SD are shown ( n = 3). * P

Techniques Used: Cell Culture, Incubation, Flow Cytometry, Cytometry, Fluorescence, Mouse Assay, Immunofluorescence, Staining, Activation Assay, Immunoprecipitation, DNA Synthesis

28) Product Images from "Transplanted endothelial cells repopulate the liver endothelium and correct the phenotype of hemophilia A mice"

Article Title: Transplanted endothelial cells repopulate the liver endothelium and correct the phenotype of hemophilia A mice

Journal: The Journal of Clinical Investigation

doi: 10.1172/JCI32748

Properties of isolated FVB/N-Tie2–GFP LSECs and fate of transplanted LSECs. Flow cytometry showing GFP-positive LSECs selected with anti-LSECs after CD45-positive cells were depleted. LSECs selected showed native GFP expression in 77% ( A ); 96% stained for the endothelial marker CD31 ( B ), and only 3% stained for CD45 ( C ). Moreover, CD31-containing LSECs coexpressed additional endothelial markers, Flk-1 ( D ) and endoglin ( E ). ( F and G ) SEM showing sieve plates with fenestrae on LSEC surface (arrowheads), another feature of sinusoidal ECs. ( H ) Donor FVB/N-Tie2–GFP mouse liver showing GFP staining (green) in LSECs, along with a portal vein radicle (asterisk). ( I – K ) FVB/N mice after LSEC transplantation. ( I ) Two transplanted cells identified by GFP staining (green) in liver sinusoids. ( J ) Increased engraftment of transplanted LSECs 1 week after cell transplantation in FLP-treated mouse. ( K ) Significantly increased engraftment of transplanted LSECs 1 week after cell transplantation in MCT-treated mouse. ( L – S ) GFP fluorescence in transplanted LSECs to indicate cell proliferation 3 months after transplantation in MCT-treated mice. ( L and P ) Nuclear staining with DAPI (blue). ( M ) Kupffer cells immunostained with F480 antibody (red). ( Q ) Endothelial cells immunostained with CD31 antibody. ( N and R ) GFP immunostaining. ( O and S ) Merged images from all 3 panels. Original magnification, ×2,500 ( F ); ×12,500 ( G ); ×200 ( H – K ); ×400 ( P – S ); ×200 ( L – O ). Scale bars: 2 μm ( F ); 0.4 μm ( G ).
Figure Legend Snippet: Properties of isolated FVB/N-Tie2–GFP LSECs and fate of transplanted LSECs. Flow cytometry showing GFP-positive LSECs selected with anti-LSECs after CD45-positive cells were depleted. LSECs selected showed native GFP expression in 77% ( A ); 96% stained for the endothelial marker CD31 ( B ), and only 3% stained for CD45 ( C ). Moreover, CD31-containing LSECs coexpressed additional endothelial markers, Flk-1 ( D ) and endoglin ( E ). ( F and G ) SEM showing sieve plates with fenestrae on LSEC surface (arrowheads), another feature of sinusoidal ECs. ( H ) Donor FVB/N-Tie2–GFP mouse liver showing GFP staining (green) in LSECs, along with a portal vein radicle (asterisk). ( I – K ) FVB/N mice after LSEC transplantation. ( I ) Two transplanted cells identified by GFP staining (green) in liver sinusoids. ( J ) Increased engraftment of transplanted LSECs 1 week after cell transplantation in FLP-treated mouse. ( K ) Significantly increased engraftment of transplanted LSECs 1 week after cell transplantation in MCT-treated mouse. ( L – S ) GFP fluorescence in transplanted LSECs to indicate cell proliferation 3 months after transplantation in MCT-treated mice. ( L and P ) Nuclear staining with DAPI (blue). ( M ) Kupffer cells immunostained with F480 antibody (red). ( Q ) Endothelial cells immunostained with CD31 antibody. ( N and R ) GFP immunostaining. ( O and S ) Merged images from all 3 panels. Original magnification, ×2,500 ( F ); ×12,500 ( G ); ×200 ( H – K ); ×400 ( P – S ); ×200 ( L – O ). Scale bars: 2 μm ( F ); 0.4 μm ( G ).

Techniques Used: Isolation, Flow Cytometry, Cytometry, Expressing, Staining, Marker, Mouse Assay, Transplantation Assay, Fluorescence, Immunostaining

29) 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

30) Product Images from "Multiserotype Protection Elicited by a Combinatorial Prime-Boost Vaccination Strategy against Bluetongue Virus"

Article Title: Multiserotype Protection Elicited by a Combinatorial Prime-Boost Vaccination Strategy against Bluetongue Virus

Journal: PLoS ONE

doi: 10.1371/journal.pone.0034735

Expression of the VP2, VP7 and NS1 BTV proteins. Immunofluorescence microscopy using a mouse polyclonal antibody against BTV-4 in BHK-21 cells transfected with pcDNA3, pcDNA3-VP2, pcDNA3-VP7, or pcDNA3-NS1 plasmids and DF-1 cells infected with rMVA, rMVA-VP2, rMVA-VP7, or rMVA-NS1.
Figure Legend Snippet: Expression of the VP2, VP7 and NS1 BTV proteins. Immunofluorescence microscopy using a mouse polyclonal antibody against BTV-4 in BHK-21 cells transfected with pcDNA3, pcDNA3-VP2, pcDNA3-VP7, or pcDNA3-NS1 plasmids and DF-1 cells infected with rMVA, rMVA-VP2, rMVA-VP7, or rMVA-NS1.

Techniques Used: Expressing, Immunofluorescence, Microscopy, Transfection, Infection

Expression of VP2, VP7, and NS1 proteins in High five cells infected with rBAC-VP2, rBAC-VP7, or rBAC-NS1. High five cells were infected with rBAC-VP2, rBAC-VP7, or rBAC-NS1 and the expression of the proteins was analysed at 24, 48 and 72 hours by western blot using a mouse polyclonal antibody against BTV-4. The position of the three proteins is indicated by an arrow.
Figure Legend Snippet: Expression of VP2, VP7, and NS1 proteins in High five cells infected with rBAC-VP2, rBAC-VP7, or rBAC-NS1. High five cells were infected with rBAC-VP2, rBAC-VP7, or rBAC-NS1 and the expression of the proteins was analysed at 24, 48 and 72 hours by western blot using a mouse polyclonal antibody against BTV-4. The position of the three proteins is indicated by an arrow.

Techniques Used: Expressing, Infection, Western Blot

Protection of VP2, VP5 and VP7 vaccinated IFNAR (−/−) mice against lethal BTV-4, BTV-8, and BTV-1 challenges. Mice (8 weeks old, 6 per group) were immunized twice by heterologous prime boost vaccination with DNAs and rMVAs expressing VP2, VP7, and NS1 BTV-4 proteins (immunized, black line) or pcDNA3 and MVA (non-immunized, dotted line), administered 2 weeks apart. Two weeks after immunization mice were intravenously inoculated with 100 PFUs (lethal dose) of BTV-8 (▪) or BTV-1 (•). (A) Survival rates of immunized and non-immunized IFNAR (−/−) mice after inoculation with BTV-8 or BTV-1. The mice were observed every 24 h for 12 days. (B) Titers of BTV-8 (▪) or BTV-1 (•) recovered in blood of immunized and non-immunized IFNAR (−/−) mice after challenge. Virus was extracted from blood and determined as described in Materials and Methods . Each point represents the mean values of the viral titer of six animals, and standard deviations are shown as error bars.
Figure Legend Snippet: Protection of VP2, VP5 and VP7 vaccinated IFNAR (−/−) mice against lethal BTV-4, BTV-8, and BTV-1 challenges. Mice (8 weeks old, 6 per group) were immunized twice by heterologous prime boost vaccination with DNAs and rMVAs expressing VP2, VP7, and NS1 BTV-4 proteins (immunized, black line) or pcDNA3 and MVA (non-immunized, dotted line), administered 2 weeks apart. Two weeks after immunization mice were intravenously inoculated with 100 PFUs (lethal dose) of BTV-8 (▪) or BTV-1 (•). (A) Survival rates of immunized and non-immunized IFNAR (−/−) mice after inoculation with BTV-8 or BTV-1. The mice were observed every 24 h for 12 days. (B) Titers of BTV-8 (▪) or BTV-1 (•) recovered in blood of immunized and non-immunized IFNAR (−/−) mice after challenge. Virus was extracted from blood and determined as described in Materials and Methods . Each point represents the mean values of the viral titer of six animals, and standard deviations are shown as error bars.

Techniques Used: Mouse Assay, Expressing

Intracellular staining of IFN-γ, in T CD8+ cells of DNA/rMVA-VP2/VP7/NS1 immunized IFNAR(−/−) mice. Two weeks after second immunization spleens were harvested and the splenocytes were stimulated with 10 µg/ml of recombinant VP2, VP7 or NS1 proteins. At 72 h post-stimulation, intracellular IFN-γ production was analysed in CD8-positive cells by flow cytometry. Results were compared with those of a non-immunized mouse. Grey bars: non-immunized mice; black bars: immunized mice. C-: unrelated stimulus, NS3 BTV protein. The results represent the average of 6 mice ± SD. Asterisks represent significant difference between samples, calculated by Man-Whitney non parametric test (p≤0.01).
Figure Legend Snippet: Intracellular staining of IFN-γ, in T CD8+ cells of DNA/rMVA-VP2/VP7/NS1 immunized IFNAR(−/−) mice. Two weeks after second immunization spleens were harvested and the splenocytes were stimulated with 10 µg/ml of recombinant VP2, VP7 or NS1 proteins. At 72 h post-stimulation, intracellular IFN-γ production was analysed in CD8-positive cells by flow cytometry. Results were compared with those of a non-immunized mouse. Grey bars: non-immunized mice; black bars: immunized mice. C-: unrelated stimulus, NS3 BTV protein. The results represent the average of 6 mice ± SD. Asterisks represent significant difference between samples, calculated by Man-Whitney non parametric test (p≤0.01).

Techniques Used: Staining, Mouse Assay, Recombinant, Flow Cytometry, Cytometry

Humoral immune response observed in IFNAR (−/−) mice vaccinated with heterologous prime boost vaccination with pcDNA3 and rMVA expressing VP2/VP7/NS1. The presence of antibodies specific of VP2, VP7, and NS1 in serum of immunized (A) and non-immunized (B) IFNAR (−/−) mice was analyzed by ELISA. Sera from mice immunized with pcDNA3-VP2,-VP7,-NS1 (day 0) and rMVA-VP2,-VP7,-NS1 (day 14) and non-immunized were collected at days 0, 14, and 28 before the challenge with BTV-4, and dilution 1∶50 was analyzed by ELISA as described in Materials and Methods . Standard deviations are shown as error bars. (C) BTV-4 neutralizing antibody detection in VP2/VP7/NS1 immunized mice by VNT. Neutralization titers at day 28 in sera of animals immunized with pcDNA3-VP2,-VP7,-NS1 (day 0) and rMVA-VP2,-VP7,-NS1 (day 14) (▪) and non-immunized (•) are shown. Means are presented (⁃) and standard deviations are shown as error bars.
Figure Legend Snippet: Humoral immune response observed in IFNAR (−/−) mice vaccinated with heterologous prime boost vaccination with pcDNA3 and rMVA expressing VP2/VP7/NS1. The presence of antibodies specific of VP2, VP7, and NS1 in serum of immunized (A) and non-immunized (B) IFNAR (−/−) mice was analyzed by ELISA. Sera from mice immunized with pcDNA3-VP2,-VP7,-NS1 (day 0) and rMVA-VP2,-VP7,-NS1 (day 14) and non-immunized were collected at days 0, 14, and 28 before the challenge with BTV-4, and dilution 1∶50 was analyzed by ELISA as described in Materials and Methods . Standard deviations are shown as error bars. (C) BTV-4 neutralizing antibody detection in VP2/VP7/NS1 immunized mice by VNT. Neutralization titers at day 28 in sera of animals immunized with pcDNA3-VP2,-VP7,-NS1 (day 0) and rMVA-VP2,-VP7,-NS1 (day 14) (▪) and non-immunized (•) are shown. Means are presented (⁃) and standard deviations are shown as error bars.

Techniques Used: Mouse Assay, Expressing, Enzyme-linked Immunosorbent Assay, Neutralization

Cytokine responses in IFNAR (−/−) mice after immunization. Sera from mice immunized with pcDNA3-VP2,-VP7,-NS1 (day 0) and rMVA-VP2,-VP7,-NS1 (day 14) (solid symbols) or pcDNA3 (day 0) and MVA (day 14) (symbols) were collected at days 0, 14, 21, and 28. The levels of cytokines were evaluated by a multiplex fluorescent bead immunoassay for quantitative detection of 5 mouse cytokines (Millipore's MILLIPLEX Mouse Cytokine kit). Samples were analyzed with a Luminex 2010 (Luminex Corporation). Means are presented (⁃).
Figure Legend Snippet: Cytokine responses in IFNAR (−/−) mice after immunization. Sera from mice immunized with pcDNA3-VP2,-VP7,-NS1 (day 0) and rMVA-VP2,-VP7,-NS1 (day 14) (solid symbols) or pcDNA3 (day 0) and MVA (day 14) (symbols) were collected at days 0, 14, 21, and 28. The levels of cytokines were evaluated by a multiplex fluorescent bead immunoassay for quantitative detection of 5 mouse cytokines (Millipore's MILLIPLEX Mouse Cytokine kit). Samples were analyzed with a Luminex 2010 (Luminex Corporation). Means are presented (⁃).

Techniques Used: Mouse Assay, Multiplex Assay, Luminex

Co-expression of the VP2, VP7 and NS1 BTV proteins. (A) Immunofluorescence microscopy using a mouse polyclonal antibody against BTV-4 in BHK-21 cells transfected with pcDNA3-VP2, pcDNA3-VP7, pcDNA3-NS1 and different combinations of these plasmids. (B) Immunofluorescence microscopy using a mouse polyclonal antibody against BTV-4, or monoclonal antibodies against VP2 and VP7 from BTV-4 in BHK-21 cells transfected with pcDNA3-VP2,-VP7,-NS1.
Figure Legend Snippet: Co-expression of the VP2, VP7 and NS1 BTV proteins. (A) Immunofluorescence microscopy using a mouse polyclonal antibody against BTV-4 in BHK-21 cells transfected with pcDNA3-VP2, pcDNA3-VP7, pcDNA3-NS1 and different combinations of these plasmids. (B) Immunofluorescence microscopy using a mouse polyclonal antibody against BTV-4, or monoclonal antibodies against VP2 and VP7 from BTV-4 in BHK-21 cells transfected with pcDNA3-VP2,-VP7,-NS1.

Techniques Used: Expressing, Immunofluorescence, Microscopy, Transfection

Protection of VP2, VP7 and NS1 vaccinated IFNAR (−/−) mice against a lethal homologous BTV-4 challenge. Mice (8 weeks old, 6 per group) were immunized twice by heterologous prime boost vaccination with pcDNA3 and rMVA expressing VP2/VP7/NS1 (▪), VP2/VP7 (▴), or NS1 (•) BTV-4 proteins (immunized, black line) or pcDNA3 and MVA (♦) (non-immunized, dotted line), administered 2 weeks apart. Two weeks after immunization all mice were intravenously inoculated with 10 3 PFUs of BTV-4 (lethal dose). Survival rates of immunized and non-immunized IFNAR (−/−) mice after inoculation with BTV-4. The mice were observed every 24 h for 12 days.
Figure Legend Snippet: Protection of VP2, VP7 and NS1 vaccinated IFNAR (−/−) mice against a lethal homologous BTV-4 challenge. Mice (8 weeks old, 6 per group) were immunized twice by heterologous prime boost vaccination with pcDNA3 and rMVA expressing VP2/VP7/NS1 (▪), VP2/VP7 (▴), or NS1 (•) BTV-4 proteins (immunized, black line) or pcDNA3 and MVA (♦) (non-immunized, dotted line), administered 2 weeks apart. Two weeks after immunization all mice were intravenously inoculated with 10 3 PFUs of BTV-4 (lethal dose). Survival rates of immunized and non-immunized IFNAR (−/−) mice after inoculation with BTV-4. The mice were observed every 24 h for 12 days.

Techniques Used: Mouse Assay, Expressing

31) Product Images from "Dendritic Cells Control CD4+CD25+ Treg Cell Suppressor Function In Vitro through Juxtacrine Delivery of IL-2"

Article Title: Dendritic Cells Control CD4+CD25+ Treg Cell Suppressor Function In Vitro through Juxtacrine Delivery of IL-2

Journal: PLoS ONE

doi: 10.1371/journal.pone.0043609

Treg suppression of the MLR-IL-2 response requires dendritic cell IL-2. Panels A–F show Treg suppression of IL-2-secreting cells in primary MLR cultures. MLR cultures of DO11.10 CD4 + CD25 − cells and allogeneic DCs from WT or IL-2 −/− KO littermates were incubated overnight with or without DO11.10 Tregs, washed, and transferred to IL-2-ELISPOT plates to measure IL-2-secreting cells as per materials and methods. A B: Tregs fail to suppress MLR-IL-2 response in cultures containing IL-2 −/− KO DCs. Bars are mean ± SE IL-2 producing cells/culture from N = 5 independent experiments with splenic DCs (A) and N = 3–4 independent experiments with BMDCs (B). * indicates p
Figure Legend Snippet: Treg suppression of the MLR-IL-2 response requires dendritic cell IL-2. Panels A–F show Treg suppression of IL-2-secreting cells in primary MLR cultures. MLR cultures of DO11.10 CD4 + CD25 − cells and allogeneic DCs from WT or IL-2 −/− KO littermates were incubated overnight with or without DO11.10 Tregs, washed, and transferred to IL-2-ELISPOT plates to measure IL-2-secreting cells as per materials and methods. A B: Tregs fail to suppress MLR-IL-2 response in cultures containing IL-2 −/− KO DCs. Bars are mean ± SE IL-2 producing cells/culture from N = 5 independent experiments with splenic DCs (A) and N = 3–4 independent experiments with BMDCs (B). * indicates p

Techniques Used: Incubation, Enzyme-linked Immunospot

Tregs preserve dendritic cell transcription of IL-2. A: DO11.10 Tregs and C57BL/6 splenic DCs were cultured together or in separate tubes for 5 and 24 h, IL-2 message was measured in cultured cells by RT-qPCR as in materials and methods. Bars are the mean ± SE of IL-2 message in the cultures. Statistical analysis was by one-way repeated measures ANOVA and Tukey's test for all pairwise multiple comparison procedure. B C: BMDCs were transfected with the IL-2 reporter plasmid (pIL2P8.4-EGFP), a negative control plasmid (pcDNA3.1/V5-His B), and a positive control plasmid to assess transfection efficiency (pAcGFP-N1). Transfectants were analyzed for IL-2-promoter activity in cells as in materials and methods. B: Panels show histograms of the level of promoter-driven GFP in gated CD11c + cells. The experiment was repeated with similar results. C: pIL-2P8.4-EGFP-transfected BMDCs were cultured unstimulated or with CpG-B, with and without Treg cells for 40 h. Bars are the mean ± SE of IL-2-promter-EGFP + BMDCs from two experiments.
Figure Legend Snippet: Tregs preserve dendritic cell transcription of IL-2. A: DO11.10 Tregs and C57BL/6 splenic DCs were cultured together or in separate tubes for 5 and 24 h, IL-2 message was measured in cultured cells by RT-qPCR as in materials and methods. Bars are the mean ± SE of IL-2 message in the cultures. Statistical analysis was by one-way repeated measures ANOVA and Tukey's test for all pairwise multiple comparison procedure. B C: BMDCs were transfected with the IL-2 reporter plasmid (pIL2P8.4-EGFP), a negative control plasmid (pcDNA3.1/V5-His B), and a positive control plasmid to assess transfection efficiency (pAcGFP-N1). Transfectants were analyzed for IL-2-promoter activity in cells as in materials and methods. B: Panels show histograms of the level of promoter-driven GFP in gated CD11c + cells. The experiment was repeated with similar results. C: pIL-2P8.4-EGFP-transfected BMDCs were cultured unstimulated or with CpG-B, with and without Treg cells for 40 h. Bars are the mean ± SE of IL-2-promter-EGFP + BMDCs from two experiments.

Techniques Used: Cell Culture, Quantitative RT-PCR, Transfection, Plasmid Preparation, Negative Control, Positive Control, Activity Assay

CD25 and cell contact dependent uptake of dendritic cell IL-2 by Treg cells. A–C: CFSE-labeled DO11.10 Tregs were stimulated with OVA peptide and different DC transfectants as in materials and methods. Panels show representative images of mCherry transfected dendritic cell (A) and IL-2mCherry transfected dendritic cell (B) in conjugate formation with Treg cell. Panels C: Three-dimensional rendering of a Treg cell localizes the IL-2mCherry to vesicles within Treg. D E: DC-Treg contact facilitates IL-2mCherry transfer from DCs to Tregs that can be blocked by anti-CD25. DO11.10 CD4 + CD25 + Treg or CD4 + CD25 − T effector (Teff) cells were cultured in triplicate in upper chambers of transwells separated from (grey bars) or in lower chambers of transwells in contact with (black bars) BMDCs from IL-2 −/− B6 mice that were transfected with IL-2mCherry (DC-IL2mCh) or control mCherry (DCmCh) and analyzed by flow cytometry as in materials and methods. Indicated cultures contained anti-CD25 antibody (antiCD25). D: Bars are the mCherry MFI means ± SE of gated Thy1.2high cells from triplicate lower chambers (black) and upper chambers (gray). E: Bars are the CD25 (left panel) and mCherry (right panel) MFI means ± SE of gated Thy1.2high cells from triplicate lower chambers (black). Statistical analysis was performed by paired t -test for within well samples and by student t-test for between well samples. Experiment was performed three times yielding similar results, representative experiments are shown.
Figure Legend Snippet: CD25 and cell contact dependent uptake of dendritic cell IL-2 by Treg cells. A–C: CFSE-labeled DO11.10 Tregs were stimulated with OVA peptide and different DC transfectants as in materials and methods. Panels show representative images of mCherry transfected dendritic cell (A) and IL-2mCherry transfected dendritic cell (B) in conjugate formation with Treg cell. Panels C: Three-dimensional rendering of a Treg cell localizes the IL-2mCherry to vesicles within Treg. D E: DC-Treg contact facilitates IL-2mCherry transfer from DCs to Tregs that can be blocked by anti-CD25. DO11.10 CD4 + CD25 + Treg or CD4 + CD25 − T effector (Teff) cells were cultured in triplicate in upper chambers of transwells separated from (grey bars) or in lower chambers of transwells in contact with (black bars) BMDCs from IL-2 −/− B6 mice that were transfected with IL-2mCherry (DC-IL2mCh) or control mCherry (DCmCh) and analyzed by flow cytometry as in materials and methods. Indicated cultures contained anti-CD25 antibody (antiCD25). D: Bars are the mCherry MFI means ± SE of gated Thy1.2high cells from triplicate lower chambers (black) and upper chambers (gray). E: Bars are the CD25 (left panel) and mCherry (right panel) MFI means ± SE of gated Thy1.2high cells from triplicate lower chambers (black). Statistical analysis was performed by paired t -test for within well samples and by student t-test for between well samples. Experiment was performed three times yielding similar results, representative experiments are shown.

Techniques Used: Labeling, Transfection, Cell Culture, Mouse Assay, Flow Cytometry, Cytometry

Dendritic cell IL-2 increases Treg phenotype. DO11.10 CD4 + CD25 + Treg cells were cultured without or with BMDCs from either WT or IL-2 −/− KO (on B6 background) mice in transwells and the level of CD25 (A) and Foxp3 (B) on Treg cells was measured by flow cytometry as in materials and methods. A: Bars are the mean ± SE of mean fluorescent intensity (MFI) of CD25 levels on Thy1.2-gated Tregs of triplicate samples from one of four representative experiments. Statistical analysis was by Tukey's test for all pairwise comparisons. B: Bars are the mean ± SE of mean fluorescent intensity (MFI) of Foxp3 levels on Thy1.2-gated Tregs of triplicate samples from a single experiment. Statistical analysis was by Tukey's test for all pairwise comparisons.
Figure Legend Snippet: Dendritic cell IL-2 increases Treg phenotype. DO11.10 CD4 + CD25 + Treg cells were cultured without or with BMDCs from either WT or IL-2 −/− KO (on B6 background) mice in transwells and the level of CD25 (A) and Foxp3 (B) on Treg cells was measured by flow cytometry as in materials and methods. A: Bars are the mean ± SE of mean fluorescent intensity (MFI) of CD25 levels on Thy1.2-gated Tregs of triplicate samples from one of four representative experiments. Statistical analysis was by Tukey's test for all pairwise comparisons. B: Bars are the mean ± SE of mean fluorescent intensity (MFI) of Foxp3 levels on Thy1.2-gated Tregs of triplicate samples from a single experiment. Statistical analysis was by Tukey's test for all pairwise comparisons.

Techniques Used: Cell Culture, Mouse Assay, Flow Cytometry, Cytometry

Tregs interfere with dendritic cell paracrine secretion of IL-2. Treg interference of dendritic cell paracrine secretion of IL-2 depends on CD25. C57BL/6 splenic DCs (left) and BMDCs (right) were cultured in IL-2 ELISPOT plates unstimulated or with CpG-B, with and without DO11.10 Treg cells, anti-CD25, or isotype control, and the number of IL-2 secreting DCs was measured as in materials and methods. Bars are the mean ± SE of IL-2 secreting DCs in triplicate samples of one of six representative experiments with splenic DCs (left) and one of three representative experiments with BMDCs (right). Statistical analysis by one-way repeated measures ANOVA and Tukey's test for all pairwise multiple comparison procedure.
Figure Legend Snippet: Tregs interfere with dendritic cell paracrine secretion of IL-2. Treg interference of dendritic cell paracrine secretion of IL-2 depends on CD25. C57BL/6 splenic DCs (left) and BMDCs (right) were cultured in IL-2 ELISPOT plates unstimulated or with CpG-B, with and without DO11.10 Treg cells, anti-CD25, or isotype control, and the number of IL-2 secreting DCs was measured as in materials and methods. Bars are the mean ± SE of IL-2 secreting DCs in triplicate samples of one of six representative experiments with splenic DCs (left) and one of three representative experiments with BMDCs (right). Statistical analysis by one-way repeated measures ANOVA and Tukey's test for all pairwise multiple comparison procedure.

Techniques Used: Cell Culture, Enzyme-linked Immunospot

32) Product Images from "Effective Non-Viral Delivery of siRNA to Acute Myeloid Leukemia Cells with Lipid-Substituted Polyethylenimines"

Article Title: Effective Non-Viral Delivery of siRNA to Acute Myeloid Leukemia Cells with Lipid-Substituted Polyethylenimines

Journal: PLoS ONE

doi: 10.1371/journal.pone.0044197

CXCR4 Silencing in THP-1 cells. Changes in CXCR4 levels based on ( A ) mean CXCR4 fluorescence intensity and ( B ) CXCR4-positive cell population. Silencing was assessed after 2 and 3 days of CXCR4-specific siRNA or control siRNA treatment (50 nM with polymer:siRNA ratio of 4∶1). The polymers used were PEI25, PEI2-LA (2.1 LA/PEI) and PEI2-CA (6.9 CA/PEI).
Figure Legend Snippet: CXCR4 Silencing in THP-1 cells. Changes in CXCR4 levels based on ( A ) mean CXCR4 fluorescence intensity and ( B ) CXCR4-positive cell population. Silencing was assessed after 2 and 3 days of CXCR4-specific siRNA or control siRNA treatment (50 nM with polymer:siRNA ratio of 4∶1). The polymers used were PEI25, PEI2-LA (2.1 LA/PEI) and PEI2-CA (6.9 CA/PEI).

Techniques Used: Fluorescence

33) Product Images from "The anti-CD74 humanized monoclonal antibody, milatuzumab, which targets the invariant chain of MHC II complexes, alters B-cell proliferation, migration, and adhesion molecule expression"

Article Title: The anti-CD74 humanized monoclonal antibody, milatuzumab, which targets the invariant chain of MHC II complexes, alters B-cell proliferation, migration, and adhesion molecule expression

Journal: Arthritis Research & Therapy

doi: 10.1186/ar3767

Surface expression of CD74, CD44, and CXCR4 on T cells, monocytes, and B cells . (A) Detection of CD74 with a commercially available FITC-labeled anti-CD74 antibody ( n = 8) and PE-labeled milatuzumab ( n = 9) on T cells, monocytes, and B cells. For each staining, representative histograms, including an isotype control or blocking experiment, are shown. Competitive blocking experiments were performed by using unlabeled milatuzumab (20-fold concentration). Significant differences were observed between the CD74 expression levels of T cells, monocytes, and B cells (Wilcoxon test), and specificity of the staining was confirmed. (B) Detection of CD74 ( n = 10), CD44 ( n = 8), and CXCR4 ( n = 12) on CD27 - naïve and CD27 + memory B cells. These surface molecules showed a distinct expression profile between these B-cell subpopulations (Wilcoxon test). ** P ≤ 0.01; *** P ≤ 0.001. BD, BD Biosciences; FITC, fluorescein isothiocyanate; MFI, (geometric) mean fluorescence intensity; PE, phycoerythrin.
Figure Legend Snippet: Surface expression of CD74, CD44, and CXCR4 on T cells, monocytes, and B cells . (A) Detection of CD74 with a commercially available FITC-labeled anti-CD74 antibody ( n = 8) and PE-labeled milatuzumab ( n = 9) on T cells, monocytes, and B cells. For each staining, representative histograms, including an isotype control or blocking experiment, are shown. Competitive blocking experiments were performed by using unlabeled milatuzumab (20-fold concentration). Significant differences were observed between the CD74 expression levels of T cells, monocytes, and B cells (Wilcoxon test), and specificity of the staining was confirmed. (B) Detection of CD74 ( n = 10), CD44 ( n = 8), and CXCR4 ( n = 12) on CD27 - naïve and CD27 + memory B cells. These surface molecules showed a distinct expression profile between these B-cell subpopulations (Wilcoxon test). ** P ≤ 0.01; *** P ≤ 0.001. BD, BD Biosciences; FITC, fluorescein isothiocyanate; MFI, (geometric) mean fluorescence intensity; PE, phycoerythrin.

Techniques Used: Expressing, Labeling, Staining, Blocking Assay, Concentration Assay, Fluorescence

34) Product Images from "The male germ cell gene regulator CTCFL is functionally different from CTCF and binds CTCF-like consensus sites in a nucleosome composition-dependent manner"

Article Title: The male germ cell gene regulator CTCFL is functionally different from CTCF and binds CTCF-like consensus sites in a nucleosome composition-dependent manner

Journal: Epigenetics & Chromatin

doi: 10.1186/1756-8935-5-8

Genome-wide analysis of CTCFL expression in ES cells. A Inducible expression of CTCFL-V5-GFP in ES cells. Notice the nuclear localization of CTCFL-V5-GFP in cells expressing the protein. B Flow chart of experiments. ES cells with a Tet-on inducible expression of a CTCFLV5-GFP transgene were sorted for GFP and used for microarray and ChIP-Seq analyses. C CTCFL expression and DNA binding are associated with elevated gene expression levels. We plotted gene expression levels, as determined by microarray analysis of induced ( ind ) or non-induced ES cells, for all genes ( all ), or those bound by CTCF, or CTCFL, to the respective promoter region (−2 k to +1 kb around TSS). Differences are highly significant ( p -value CTCF-ind versus CTCFL-ind: 5.1 × e -14 ; p -value CTCF versus CTCFL: 5.9 × e -13 ). D Transcript analyses in ES cells expressing CTCFL-V5-GFP. Real-time RT-PCR expression analyses of CTCFL-V5-GFP-induced and GFP-sorted ES cells, relative to non-induced ES cells, for the indicated genes, referenced to Cdk2 expression. E Venn diagram of DNA-binding sites for CTCFL and CTCF. F Clustered heatmap representation of three classes of CTCF/CTCFL-binding sites. Shown are the binding profiles of CTCFL and CTCF ( 1 : our own data; 2 : [ 7 ]) across all CTCF/CTCFL-binding sites identified in mES cells. Sites are grouped into CTCFL-only, CTCF-only, and combined CTCFL and CTCF sites. Within the three classes, data sets were sorted decreasingly from top to bottom for average binding across the interval from 2 kb to +2 kb around the identified binding peak center positions. Additionally the occurrences of predicted CTCFL motifs within these intervals are plotted. G Motif comparison of CTCF and CTCFL. DNA-binding motif for CTCFL-only ( top panel ), CTCF + CTCFL ( middle panel ) and CTCF-only binding sites ( bottom panel ).
Figure Legend Snippet: Genome-wide analysis of CTCFL expression in ES cells. A Inducible expression of CTCFL-V5-GFP in ES cells. Notice the nuclear localization of CTCFL-V5-GFP in cells expressing the protein. B Flow chart of experiments. ES cells with a Tet-on inducible expression of a CTCFLV5-GFP transgene were sorted for GFP and used for microarray and ChIP-Seq analyses. C CTCFL expression and DNA binding are associated with elevated gene expression levels. We plotted gene expression levels, as determined by microarray analysis of induced ( ind ) or non-induced ES cells, for all genes ( all ), or those bound by CTCF, or CTCFL, to the respective promoter region (−2 k to +1 kb around TSS). Differences are highly significant ( p -value CTCF-ind versus CTCFL-ind: 5.1 × e -14 ; p -value CTCF versus CTCFL: 5.9 × e -13 ). D Transcript analyses in ES cells expressing CTCFL-V5-GFP. Real-time RT-PCR expression analyses of CTCFL-V5-GFP-induced and GFP-sorted ES cells, relative to non-induced ES cells, for the indicated genes, referenced to Cdk2 expression. E Venn diagram of DNA-binding sites for CTCFL and CTCF. F Clustered heatmap representation of three classes of CTCF/CTCFL-binding sites. Shown are the binding profiles of CTCFL and CTCF ( 1 : our own data; 2 : [ 7 ]) across all CTCF/CTCFL-binding sites identified in mES cells. Sites are grouped into CTCFL-only, CTCF-only, and combined CTCFL and CTCF sites. Within the three classes, data sets were sorted decreasingly from top to bottom for average binding across the interval from 2 kb to +2 kb around the identified binding peak center positions. Additionally the occurrences of predicted CTCFL motifs within these intervals are plotted. G Motif comparison of CTCF and CTCFL. DNA-binding motif for CTCFL-only ( top panel ), CTCF + CTCFL ( middle panel ) and CTCF-only binding sites ( bottom panel ).

Techniques Used: Genome Wide, Expressing, Flow Cytometry, Microarray, Chromatin Immunoprecipitation, Binding Assay, Quantitative RT-PCR

CTCFL is functionally different from CTCF A Strategy for the rescue of CTCF-depleted ES cells. Ctcf lox/lox ES cells were infected with lentivirus containing the Cre recombinase and/or fluorescently tagged CTCF(L) proteins. After infection neomycin-resistant colonies were picked and analyzed. m = mouse, g = chicken. B Analysis of Ctcf lox/lox deletion. After infection with CRE-containing constructs, Ctcf lox/lox ES cells were scored for the status of the conditional Ctcf alleles by DNA blot. The position of wild-type (wt), deleted (Ctcf del , or del) and conditional (Ctcf lox , or lox) loci in control ES cells ( C ), non-treated Ctcf lox/lox ES cells ( 1 ) and lentivirally transduced Ctcf lox/lox ES cells ( 2 – 5 , see panel A for numbering of constructs) is indicated. Cells are considered rescued when both conditional CTCF alleles have been deleted. C Analysis of CTCF protein expression. Neomycin-resistant colonies were grown and analyzed by Western blot for CTCF ( upper panel ) or GFP ( lower panel ) expression. Note that rescued cells are negative for endogenous CTCF. D , E GFP-CTCFL is a functional protein. ES cells were transiently transfected and harvested after 1 day. ChIP (DNA, D ) and RT-PCR (mRNA, E ) analyses revealed that GFP-CTCFL binds Vps18, Stra8 and Prss50 promoters ( D ) and is able to induce expression of Gal3st1, Stra8 and Prss50 ( E ).
Figure Legend Snippet: CTCFL is functionally different from CTCF A Strategy for the rescue of CTCF-depleted ES cells. Ctcf lox/lox ES cells were infected with lentivirus containing the Cre recombinase and/or fluorescently tagged CTCF(L) proteins. After infection neomycin-resistant colonies were picked and analyzed. m = mouse, g = chicken. B Analysis of Ctcf lox/lox deletion. After infection with CRE-containing constructs, Ctcf lox/lox ES cells were scored for the status of the conditional Ctcf alleles by DNA blot. The position of wild-type (wt), deleted (Ctcf del , or del) and conditional (Ctcf lox , or lox) loci in control ES cells ( C ), non-treated Ctcf lox/lox ES cells ( 1 ) and lentivirally transduced Ctcf lox/lox ES cells ( 2 – 5 , see panel A for numbering of constructs) is indicated. Cells are considered rescued when both conditional CTCF alleles have been deleted. C Analysis of CTCF protein expression. Neomycin-resistant colonies were grown and analyzed by Western blot for CTCF ( upper panel ) or GFP ( lower panel ) expression. Note that rescued cells are negative for endogenous CTCF. D , E GFP-CTCFL is a functional protein. ES cells were transiently transfected and harvested after 1 day. ChIP (DNA, D ) and RT-PCR (mRNA, E ) analyses revealed that GFP-CTCFL binds Vps18, Stra8 and Prss50 promoters ( D ) and is able to induce expression of Gal3st1, Stra8 and Prss50 ( E ).

Techniques Used: Infection, Construct, Expressing, Western Blot, Functional Assay, Transfection, Chromatin Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction

Characterization of CTCF and CTCFL binding. A Examples of CTCF- and CTCFL-binding site location. The genomic location of CTCF ( upper part ) and CTCFL ( middle and bottom parts) binding sites in the absence (−CTCFL, middle ) or presence (+CTCFL, bottom ) of CTCFL, within the Stra8, Prss50 and Gal3st1 genes. The vertical axes show the number of unique sequence reads. B CTCFL binds to Stra8 and Prss50. Band shift analyses of GFP-CTCFL on Stra8 and Prss50 fragments. GFP-CTCFL binding can be super shifted (marked with asterisks ) with anti-GFP, but not with an Actin antibody. Band shifts were performed under excess probe conditions. C In vitro effect of CTCFL on CTCF binding. Band shift analyses with GFP-CTCF and/or GFP-CTCFL on Prss50- and Stra8-bindings sites. GFP-CTCFL is added in increasing amounts (1-, 2-, 5- and 10-fold compared to GFP-CTCF). To allow competition, the band shift was performed under probe-limiting concentrations. D Cellular effect of CTCFL on CTCF binding. ChIP analyses with CTCFL ( blue ), CTCF ( red ) and pre-immune ( yellow ) antisera in ES cells that were either non-transfected (−) or transiently transfected CTCFL-V5-GFP (+). According to ChIP-sequencing data, Prss50, Stra8 and Vps18 bind both CTCF and CTCFL, whereas Gal3st1 only binds CTCFL, and Chr10 only binds CTCF. A CTCF- and CTCFL-negative site within the Amylase gene is used as reference and set to 1. Error bars represent standard deviations of biological replicates. E Competition between CTCF and CTCFL in ES cells. Genome-wide binding of CTCF was compared to that of CTCFL by ChIP-Seq using non-transfected ES cells and ES cells transiently transfected with GFP-CTCFL. The left hand panel shows the effect of CTCFL binding on shared CTCFL/CTCF sites that showed > 1.5 fold difference in CTCF binding. The effect is categorized into sites with increased ( up ) or decreased ( down ) CTCF binding. The right hand panel shows a more general effect of CTCFL binding on CTCF binding. Here, we examined the change in CTCF binding in all shared CTCF(L)-binding sites ( all ) compared to those shared sites that were significantly changed in CTCF binding ( changed ). The effect on CTCF binding is plotted as log2-fold difference. F In vivo CTCF(L) binding. ChIP was performed using anti-CTCF ( red ) or anti-CTCFL ( blue ) antibodies, or pre-immune serum, on the indicated sites ( A : Amylase, S : Stra8, P : Prss50, V : Vps18) in nuclei from dissociated seminiferous tubules, partly purified by elutriation. Relative enrichment is shown compared to Amylase.
Figure Legend Snippet: Characterization of CTCF and CTCFL binding. A Examples of CTCF- and CTCFL-binding site location. The genomic location of CTCF ( upper part ) and CTCFL ( middle and bottom parts) binding sites in the absence (−CTCFL, middle ) or presence (+CTCFL, bottom ) of CTCFL, within the Stra8, Prss50 and Gal3st1 genes. The vertical axes show the number of unique sequence reads. B CTCFL binds to Stra8 and Prss50. Band shift analyses of GFP-CTCFL on Stra8 and Prss50 fragments. GFP-CTCFL binding can be super shifted (marked with asterisks ) with anti-GFP, but not with an Actin antibody. Band shifts were performed under excess probe conditions. C In vitro effect of CTCFL on CTCF binding. Band shift analyses with GFP-CTCF and/or GFP-CTCFL on Prss50- and Stra8-bindings sites. GFP-CTCFL is added in increasing amounts (1-, 2-, 5- and 10-fold compared to GFP-CTCF). To allow competition, the band shift was performed under probe-limiting concentrations. D Cellular effect of CTCFL on CTCF binding. ChIP analyses with CTCFL ( blue ), CTCF ( red ) and pre-immune ( yellow ) antisera in ES cells that were either non-transfected (−) or transiently transfected CTCFL-V5-GFP (+). According to ChIP-sequencing data, Prss50, Stra8 and Vps18 bind both CTCF and CTCFL, whereas Gal3st1 only binds CTCFL, and Chr10 only binds CTCF. A CTCF- and CTCFL-negative site within the Amylase gene is used as reference and set to 1. Error bars represent standard deviations of biological replicates. E Competition between CTCF and CTCFL in ES cells. Genome-wide binding of CTCF was compared to that of CTCFL by ChIP-Seq using non-transfected ES cells and ES cells transiently transfected with GFP-CTCFL. The left hand panel shows the effect of CTCFL binding on shared CTCFL/CTCF sites that showed > 1.5 fold difference in CTCF binding. The effect is categorized into sites with increased ( up ) or decreased ( down ) CTCF binding. The right hand panel shows a more general effect of CTCFL binding on CTCF binding. Here, we examined the change in CTCF binding in all shared CTCF(L)-binding sites ( all ) compared to those shared sites that were significantly changed in CTCF binding ( changed ). The effect on CTCF binding is plotted as log2-fold difference. F In vivo CTCF(L) binding. ChIP was performed using anti-CTCF ( red ) or anti-CTCFL ( blue ) antibodies, or pre-immune serum, on the indicated sites ( A : Amylase, S : Stra8, P : Prss50, V : Vps18) in nuclei from dissociated seminiferous tubules, partly purified by elutriation. Relative enrichment is shown compared to Amylase.

Techniques Used: Binding Assay, Sequencing, Electrophoretic Mobility Shift Assay, In Vitro, Chromatin Immunoprecipitation, Transfection, Genome Wide, In Vivo, Purification

Expression of CTCFL and CTCF in the testis. A - C Immunohistochemical staining of testis sections. Paraffin-embedded sections from day 90 testes from heterozygous (del/+) and homozygous (del/del) Ctcfl mutant mice were stained with anti-CTCFL, followed by diaminobenzidine ( DAB ) coloring. Some of the CTCFL-positive cells are indicated with black arrowheads . Scale bars A , C : 100 μm, B : 50 μm. D - G Immunofluorescence staining of testis sections. Sections as described in A - C were stained with CTCFL ( D and F ) or STRA8 ( E and G ) antibodies. STRA8-positive cells in panels E and G are indicated with green arrowheads ; the same cells are indicated with red arrowheads in the sections stained with anti-CTCFL antibodies (panels D and F ). In Ctcfl mutant mice, STRA8 distribution is not changed. Scale bar is 50 μm. H - P Ex vivo confocal and multiphoton imaging of intact seminiferous tubules. Testis tubules were dissected from GFP-CTCFL- ( H - M ) or GFP-CTCF- ( N - P ) expressing mice, exposed to Hoechst at the adluminal side of the seminiferous tubule, and analyzed with a confocal/multiphoton microscope (GFP-CTCFL and GFP-CTCF, green ; Hoechst, red ). Panel H - J (see also Movie S1) shows a low magnification view of GFP-CTCFL distribution. Notice the presence of GFP-CTCFL-positive cells in the upper half of the tubule and their absence in the bottom half, indicating a transient population of cells. In ( K - M ) a high-magnification view of the same GFP-CTCFL-positive cells is shown. Notice the non-homogenous distribution of GFP-CTCFL in the nucleus. In ( N - P ) GFP-CTCF staining is shown. For clarity, some of the cell types are encircled, and their position is indicated in the other panels using white arrowheads . Pl = preleptone spermatocyte; rs = round spermatid; pa = pachytene spermatocyte; se = Sertoli cell. Bars, H - J : 70 μm, K - M : 10 μm, N - P : 25 μm.
Figure Legend Snippet: Expression of CTCFL and CTCF in the testis. A - C Immunohistochemical staining of testis sections. Paraffin-embedded sections from day 90 testes from heterozygous (del/+) and homozygous (del/del) Ctcfl mutant mice were stained with anti-CTCFL, followed by diaminobenzidine ( DAB ) coloring. Some of the CTCFL-positive cells are indicated with black arrowheads . Scale bars A , C : 100 μm, B : 50 μm. D - G Immunofluorescence staining of testis sections. Sections as described in A - C were stained with CTCFL ( D and F ) or STRA8 ( E and G ) antibodies. STRA8-positive cells in panels E and G are indicated with green arrowheads ; the same cells are indicated with red arrowheads in the sections stained with anti-CTCFL antibodies (panels D and F ). In Ctcfl mutant mice, STRA8 distribution is not changed. Scale bar is 50 μm. H - P Ex vivo confocal and multiphoton imaging of intact seminiferous tubules. Testis tubules were dissected from GFP-CTCFL- ( H - M ) or GFP-CTCF- ( N - P ) expressing mice, exposed to Hoechst at the adluminal side of the seminiferous tubule, and analyzed with a confocal/multiphoton microscope (GFP-CTCFL and GFP-CTCF, green ; Hoechst, red ). Panel H - J (see also Movie S1) shows a low magnification view of GFP-CTCFL distribution. Notice the presence of GFP-CTCFL-positive cells in the upper half of the tubule and their absence in the bottom half, indicating a transient population of cells. In ( K - M ) a high-magnification view of the same GFP-CTCFL-positive cells is shown. Notice the non-homogenous distribution of GFP-CTCFL in the nucleus. In ( N - P ) GFP-CTCF staining is shown. For clarity, some of the cell types are encircled, and their position is indicated in the other panels using white arrowheads . Pl = preleptone spermatocyte; rs = round spermatid; pa = pachytene spermatocyte; se = Sertoli cell. Bars, H - J : 70 μm, K - M : 10 μm, N - P : 25 μm.

Techniques Used: Expressing, Immunohistochemistry, Staining, Mutagenesis, Mouse Assay, Immunofluorescence, Ex Vivo, Imaging, Microscopy

Ctcfl and Ctcf expression and targeting. A B RNAse protection analysis of Ctcfl and Ctcf. For Ctcfl ( A ) RNase protection analysis (RPA) was performed on polyA purified mRNA with probes covering parts of Ctcfl exon 8 and 9 ( left , small fragment) or a 5’end RACE product ( right , large fragment). For Ctcf ( B ) the RPA was performed on total RNA with probes protecting Ctcf exon 2. The positions of the respective protected fragments are indicated with arrows . Ctcfl mRNA mRNA can only be detected in polyA purified mRNA from testis ( t ), whereas Ctcf is identified in total RNA from all three tissues tested. M , marker; p , input probe; c , tRNA control; h , heart; t , testis; b , brain. Aprt exon 3 is used as loading control and marked by an asterisk [ 28 ]. This analysis identifies the first exon containing the ATG translation initiation codon in Ctcfl and shows that Ctcfl is expressed in testis. C Schematic overview of the modified Ctcfl alleles and targeting constructs. The Ctcfl locus is shown on scale, with the constructs (not on scale) used for homologous recombination in ES cells underneath. Targeting at the 5’end of Ctcfl yielded the Ctcfl gpf- neo allele. Cre-mediated excision of the LoxP-embedded neomcyin resistance gene yielded the Ctcfl gfp allele (not shown). The 3’end targeting was performed on the Ctcfl gpf- neo allele, and yielded the Ctcfl gfp -neo-puro allele. Cre-mediated excision of the sequence in between the outermost LoxP sites yielded the Ctcfl del allele, in which exons 1–8 of the Ctcfl gene are deleted (not shown). A major difference between the Ctcfl del allele described here and the Ctcfl knockout published earlier [ 26 ] is that in the Ctcfl del allele the GFP coding sequence is fused in frame with the CTCFL coding sequence. Black boxes represent exons, GFP tag, neomycin and puromycin cassettes. Probes a, b, c, d and e are indicated by lines . Oligos 1, 2, 3 and 4 are represented by arrowheads . White triangles are LoxP sites. B = BglII; N = NcoI; S = SpeI; A = AvrII. D DNA blot showing Ctcfl targeting. Probes a and b were used on DNA blots from ES cells for identification of the Ctcfl gfp -neo allele and probes c and d for the Ctcfl puro allele. Probe e identifies the Ctcfl del allele from Ctcfl gfp -neo-puro mice that were crossed to a chicken Actin-Cre transgene. Probe a, HindIII digest (wt 8.9 kb, ko 5.7 kb); probe b, EcoRI digest (wt 14 kb; ko 11 kb); probe c, BamHI digest (wt 16.1 kb; ko 6.8 kb); probe d, BamHI digest (wt 16.1 kb; ko 11.1 kb). E Absence of Ctcfl DNA in the Ctcfl del allele. PCR on tail DNA indicates that Ctcfl del/del mice are deleted for exons 1–8 ( top three panels ) and are positive for GFP (oligos 2 and 4). F Absence of Ctcfl RNA in Ctcfl mutant mice. PCR on cDNA derived from testis mRNA shows that Ctcfl is absent from Ctcfl del/del mice. Acrosin and Gapd function as positive controls. G Schematic overview of the Ctcf allele and targeting strategy for the Ctcf gfp-neo allele. The Ctcf locus is shown on scale, with the construct (not on scale) used for homologous recombination in ES cells underneath. Cre-mediated excision of the LoxP-embedded neomcyin resistance gene yielded the Ctcf gfp (or Ctcf ki ) allele (not shown). Black boxes represent exons, GFP tag and neomycin cassette. Oligos 5, 6, 7 and 8 are represented by arrowheads . White triangles are LoxP sites. E = EcoRI. H PCR confirming Ctcf gpf-neo allele. Identification of the CTCF gfp-neo (or Ctcf ki ) allele by PCR with oligos 7 and 8 or oligos 5, 6 and 8 (see panel G ). I Western blot confirming GFP-CTCF expression from the Ctcf gfp allele. We isolated MEFS from E13.5 day wild-type (+/+), heterozygous Ctcf gfp/+ (or Ctcf ki/+ ) or homozygous Ctcf gfp/gfp (or Ctcf ki/ki ) embryos, and identified the GFP-CTCF fusion protein by Western blot of MEF extracts using anti-CTCF or anti-GFP antibodies. Note the increased size of the GFP-CTCF protein compared to the CTCF protein due to the GFP tag.
Figure Legend Snippet: Ctcfl and Ctcf expression and targeting. A B RNAse protection analysis of Ctcfl and Ctcf. For Ctcfl ( A ) RNase protection analysis (RPA) was performed on polyA purified mRNA with probes covering parts of Ctcfl exon 8 and 9 ( left , small fragment) or a 5’end RACE product ( right , large fragment). For Ctcf ( B ) the RPA was performed on total RNA with probes protecting Ctcf exon 2. The positions of the respective protected fragments are indicated with arrows . Ctcfl mRNA mRNA can only be detected in polyA purified mRNA from testis ( t ), whereas Ctcf is identified in total RNA from all three tissues tested. M , marker; p , input probe; c , tRNA control; h , heart; t , testis; b , brain. Aprt exon 3 is used as loading control and marked by an asterisk [ 28 ]. This analysis identifies the first exon containing the ATG translation initiation codon in Ctcfl and shows that Ctcfl is expressed in testis. C Schematic overview of the modified Ctcfl alleles and targeting constructs. The Ctcfl locus is shown on scale, with the constructs (not on scale) used for homologous recombination in ES cells underneath. Targeting at the 5’end of Ctcfl yielded the Ctcfl gpf- neo allele. Cre-mediated excision of the LoxP-embedded neomcyin resistance gene yielded the Ctcfl gfp allele (not shown). The 3’end targeting was performed on the Ctcfl gpf- neo allele, and yielded the Ctcfl gfp -neo-puro allele. Cre-mediated excision of the sequence in between the outermost LoxP sites yielded the Ctcfl del allele, in which exons 1–8 of the Ctcfl gene are deleted (not shown). A major difference between the Ctcfl del allele described here and the Ctcfl knockout published earlier [ 26 ] is that in the Ctcfl del allele the GFP coding sequence is fused in frame with the CTCFL coding sequence. Black boxes represent exons, GFP tag, neomycin and puromycin cassettes. Probes a, b, c, d and e are indicated by lines . Oligos 1, 2, 3 and 4 are represented by arrowheads . White triangles are LoxP sites. B = BglII; N = NcoI; S = SpeI; A = AvrII. D DNA blot showing Ctcfl targeting. Probes a and b were used on DNA blots from ES cells for identification of the Ctcfl gfp -neo allele and probes c and d for the Ctcfl puro allele. Probe e identifies the Ctcfl del allele from Ctcfl gfp -neo-puro mice that were crossed to a chicken Actin-Cre transgene. Probe a, HindIII digest (wt 8.9 kb, ko 5.7 kb); probe b, EcoRI digest (wt 14 kb; ko 11 kb); probe c, BamHI digest (wt 16.1 kb; ko 6.8 kb); probe d, BamHI digest (wt 16.1 kb; ko 11.1 kb). E Absence of Ctcfl DNA in the Ctcfl del allele. PCR on tail DNA indicates that Ctcfl del/del mice are deleted for exons 1–8 ( top three panels ) and are positive for GFP (oligos 2 and 4). F Absence of Ctcfl RNA in Ctcfl mutant mice. PCR on cDNA derived from testis mRNA shows that Ctcfl is absent from Ctcfl del/del mice. Acrosin and Gapd function as positive controls. G Schematic overview of the Ctcf allele and targeting strategy for the Ctcf gfp-neo allele. The Ctcf locus is shown on scale, with the construct (not on scale) used for homologous recombination in ES cells underneath. Cre-mediated excision of the LoxP-embedded neomcyin resistance gene yielded the Ctcf gfp (or Ctcf ki ) allele (not shown). Black boxes represent exons, GFP tag and neomycin cassette. Oligos 5, 6, 7 and 8 are represented by arrowheads . White triangles are LoxP sites. E = EcoRI. H PCR confirming Ctcf gpf-neo allele. Identification of the CTCF gfp-neo (or Ctcf ki ) allele by PCR with oligos 7 and 8 or oligos 5, 6 and 8 (see panel G ). I Western blot confirming GFP-CTCF expression from the Ctcf gfp allele. We isolated MEFS from E13.5 day wild-type (+/+), heterozygous Ctcf gfp/+ (or Ctcf ki/+ ) or homozygous Ctcf gfp/gfp (or Ctcf ki/ki ) embryos, and identified the GFP-CTCF fusion protein by Western blot of MEF extracts using anti-CTCF or anti-GFP antibodies. Note the increased size of the GFP-CTCF protein compared to the CTCF protein due to the GFP tag.

Techniques Used: Expressing, Recombinase Polymerase Amplification, Purification, Marker, Modification, Construct, Homologous Recombination, Sequencing, Knock-Out, Mouse Assay, Polymerase Chain Reaction, Mutagenesis, Derivative Assay, Western Blot, Isolation

35) Product Images from "Role of Murine Intestinal Interleukin-1 Receptor 1-Expressing Lymphoid Tissue Inducer-Like Cells in Salmonella Infection"

Article Title: Role of Murine Intestinal Interleukin-1 Receptor 1-Expressing Lymphoid Tissue Inducer-Like Cells in Salmonella Infection

Journal: PLoS ONE

doi: 10.1371/journal.pone.0065405

Colonic LTi-like cells are significant innate producers of IL-22. (A) Representative FACS histogram depicting percentage of CD4 + LTi-like cells that produce IL-22 under control conditions ( left panel ) and in DSS colitis ( right panel ) in Rag1 −/− C57BL/6J mice. (B) Graph summarizing percentage of CD4 + LTi-like cells producing IL-22. (C) Representative scatter plot depicting the phenotype of IL-22-producing lymphocytes under control conditions ( left panel ) and in DSS colitis ( right panel ) in Rag1 −/− C57BL/6J mice. Gated on IL-22 + lymphocytes. (D) Graph depicting percentage of IL-22-producing lymphocytes that are CD4 + LTi-like cells. All graphs represent three independent experiments. *, p
Figure Legend Snippet: Colonic LTi-like cells are significant innate producers of IL-22. (A) Representative FACS histogram depicting percentage of CD4 + LTi-like cells that produce IL-22 under control conditions ( left panel ) and in DSS colitis ( right panel ) in Rag1 −/− C57BL/6J mice. (B) Graph summarizing percentage of CD4 + LTi-like cells producing IL-22. (C) Representative scatter plot depicting the phenotype of IL-22-producing lymphocytes under control conditions ( left panel ) and in DSS colitis ( right panel ) in Rag1 −/− C57BL/6J mice. Gated on IL-22 + lymphocytes. (D) Graph depicting percentage of IL-22-producing lymphocytes that are CD4 + LTi-like cells. All graphs represent three independent experiments. *, p

Techniques Used: FACS, Mouse Assay

IL-1R1 is required for IL-23-stimulated IL-17 and IL-22 production by LTi-like cells in vitro . (A and B) Box and whiskers plot depicting percent of WT (W) or IL-1R1 −/− (I) colonic CD4 + LTi-like cells that produce IL-22 (A) or IL-17 (B). (C) Box and whiskers plot depicting percent of colonic LTi-like cells isolated from Rag1 −/− (R) C57BL/6J mice that produce IL-22. (D) Box and whiskers plot depicting percent of WT (W) or IL-1R1 −/− (I) colonic CD4 + LTi-like cells that produce IFN-γ. Except in (C), cells were isolated from WT ( top panels ) or IL-1R1 −/− C57BL/6J mice ( bottom panels ). Cells were stimulated by rIL-23 (23; right panels ) or medium (M; left panels ). Box and whisker plots representative of at least three independent experiments. *, p
Figure Legend Snippet: IL-1R1 is required for IL-23-stimulated IL-17 and IL-22 production by LTi-like cells in vitro . (A and B) Box and whiskers plot depicting percent of WT (W) or IL-1R1 −/− (I) colonic CD4 + LTi-like cells that produce IL-22 (A) or IL-17 (B). (C) Box and whiskers plot depicting percent of colonic LTi-like cells isolated from Rag1 −/− (R) C57BL/6J mice that produce IL-22. (D) Box and whiskers plot depicting percent of WT (W) or IL-1R1 −/− (I) colonic CD4 + LTi-like cells that produce IFN-γ. Except in (C), cells were isolated from WT ( top panels ) or IL-1R1 −/− C57BL/6J mice ( bottom panels ). Cells were stimulated by rIL-23 (23; right panels ) or medium (M; left panels ). Box and whisker plots representative of at least three independent experiments. *, p

Techniques Used: In Vitro, Isolation, Mouse Assay, Whisker Assay

36) Product Images from "Interleukin-33 amplifies IgE synthesis and triggers mast cell degranulation via interleukin-4 in na?ve mice 1"

Article Title: Interleukin-33 amplifies IgE synthesis and triggers mast cell degranulation via interleukin-4 in na?ve mice 1

Journal: Allergy

doi: 10.1111/j.1398-9995.2012.02859.x

Schematic representation of the mechanism of IL-33-induced IgE synthesis and anaphylaxis in naive mice. IL-33 binding to ST2 + innate cells, such as mast cells and eosinophils, leads to mast cell proliferation and increased IL-4 synthesis. IL-4 would then activate B cells to proliferate and to produce IgE. IL-4 produced by the innate cells could also activate T cells to express CD40L that interacts with CD40 on B cells to further enhance IgE production. IgE, together with IL-33 and IL-4, would stimulate mast cells to degranulate, resulting in anaphylaxis.
Figure Legend Snippet: Schematic representation of the mechanism of IL-33-induced IgE synthesis and anaphylaxis in naive mice. IL-33 binding to ST2 + innate cells, such as mast cells and eosinophils, leads to mast cell proliferation and increased IL-4 synthesis. IL-4 would then activate B cells to proliferate and to produce IgE. IL-4 produced by the innate cells could also activate T cells to express CD40L that interacts with CD40 on B cells to further enhance IgE production. IgE, together with IL-33 and IL-4, would stimulate mast cells to degranulate, resulting in anaphylaxis.

Techniques Used: Mouse Assay, Binding Assay, Produced

IL-4Rα T cells contribute to IL-33-induced IgE synthesis. Wild-type (WT), IL-4 −/− or T-IL-4Rα −/− mice were treated with IL-33. (A) Serum IgE and (B) IL-4 and IL-13 concentrations were measured by ELISA. (C) The levels of CD40L and CD25 on CD4 + T cells and the number of CD4 + CD40L + , CD4 + CD25 + T cells in the spleen of WT and IL-4 −/− mice were determined by FACScan and differential counting. (D) Total CD4 + T and CD19 + B cells in the spleen of WT and IL-4 −/− mice were determined by differential cell count. Data are from two experiments, n = 6 mice per group, * P
Figure Legend Snippet: IL-4Rα T cells contribute to IL-33-induced IgE synthesis. Wild-type (WT), IL-4 −/− or T-IL-4Rα −/− mice were treated with IL-33. (A) Serum IgE and (B) IL-4 and IL-13 concentrations were measured by ELISA. (C) The levels of CD40L and CD25 on CD4 + T cells and the number of CD4 + CD40L + , CD4 + CD25 + T cells in the spleen of WT and IL-4 −/− mice were determined by FACScan and differential counting. (D) Total CD4 + T and CD19 + B cells in the spleen of WT and IL-4 −/− mice were determined by differential cell count. Data are from two experiments, n = 6 mice per group, * P

Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Cell Counting

37) Product Images from "Adhesive Signature-based, Label-free Isolation of Human Pluripotent Stem Cells"

Article Title: Adhesive Signature-based, Label-free Isolation of Human Pluripotent Stem Cells

Journal: Nature methods

doi: 10.1038/nmeth.2437

μSHEAR-based isolation of bona fide hiPSCs from a heterogeneous reprogramming culture ( a ) A heterogeneous reprogramming culture seeded into μSHEAR device. Colonies of hiPSCs were selectively detached within 5 min of flow at 100 dynes cm −2 shear stress. ( b ) Flow cytometry plots showing detached hiPSCs (TRA-1-60 + CMPTX + ) and non-reprogrammed/partially reprogrammed cells (TRA-1-60 − CMPTX + ). At 100 dynes cm −2 shear stress, hiPSCs were isolated with 95% purity from a heterogeneous reprogramming culture with initial 0.65% hiPSC purity. Following μSHEAR isolation, residual cells in the devices were trypsinized and the residual cells consisted of 99.9% non-hiPSCs. μSHEAR-isolated hiPSCs and residual culture from the devices were re-plated on Matrigel and stained for bona fide hiPSC markers for fully reprogrammed cells: ( c ) OCT4 and TRA-1-81. Isolated hiPSCs expressed both markers whereas residual cells contained several cells expressing OCT4 but negative for TRA-1-81. ( d ) Merged phase contrast and OCT4, TRA-1-81 staining for the residual cells indicate presence of OCT4 + TRA-1-80 − cells with elongated and spread morphology, distinct from hiPSCs. ( e–h ) NANOG, TRA-1-60, REX1, SSEA4, hTERT, GDF, and DNM3Tb. Isolated hiPSCs expressed all bona fide markers. Residual cells were negative for NANOG, TRA-1-81, REX1, SSEA4, and DNMT3b but expressed hTERT and GDF. ( i ) Representative hematoxylin-eosin (H/E) stained sections from a formalin-fixed teratoma produced from μSHEAR-isolated hiPSCs. μSHEAR-isolated formed differentiated tissues representing all three embryonic germ layers including: cartilage (mesoderm), glandular epithelium (endoderm), and neural tissues (ectoderm).
Figure Legend Snippet: μSHEAR-based isolation of bona fide hiPSCs from a heterogeneous reprogramming culture ( a ) A heterogeneous reprogramming culture seeded into μSHEAR device. Colonies of hiPSCs were selectively detached within 5 min of flow at 100 dynes cm −2 shear stress. ( b ) Flow cytometry plots showing detached hiPSCs (TRA-1-60 + CMPTX + ) and non-reprogrammed/partially reprogrammed cells (TRA-1-60 − CMPTX + ). At 100 dynes cm −2 shear stress, hiPSCs were isolated with 95% purity from a heterogeneous reprogramming culture with initial 0.65% hiPSC purity. Following μSHEAR isolation, residual cells in the devices were trypsinized and the residual cells consisted of 99.9% non-hiPSCs. μSHEAR-isolated hiPSCs and residual culture from the devices were re-plated on Matrigel and stained for bona fide hiPSC markers for fully reprogrammed cells: ( c ) OCT4 and TRA-1-81. Isolated hiPSCs expressed both markers whereas residual cells contained several cells expressing OCT4 but negative for TRA-1-81. ( d ) Merged phase contrast and OCT4, TRA-1-81 staining for the residual cells indicate presence of OCT4 + TRA-1-80 − cells with elongated and spread morphology, distinct from hiPSCs. ( e–h ) NANOG, TRA-1-60, REX1, SSEA4, hTERT, GDF, and DNM3Tb. Isolated hiPSCs expressed all bona fide markers. Residual cells were negative for NANOG, TRA-1-81, REX1, SSEA4, and DNMT3b but expressed hTERT and GDF. ( i ) Representative hematoxylin-eosin (H/E) stained sections from a formalin-fixed teratoma produced from μSHEAR-isolated hiPSCs. μSHEAR-isolated formed differentiated tissues representing all three embryonic germ layers including: cartilage (mesoderm), glandular epithelium (endoderm), and neural tissues (ectoderm).

Techniques Used: Isolation, Flow Cytometry, Cytometry, Staining, Expressing, Produced

Adhesive differences in spontaneously differentiated pluripotent stem cells enable adhesive force-based enrichment of undifferentiated cells ( a ) μSHEAR-based isolation of UD-hiPSCs (white arrowhead) from SD-hiPSCs (red arrowhead) at 100 dynes cm −2 with high enrichment efficiency irrespective of SD-hiPSC contamination (table). Bottom panel shows non-selective detachment of cells using a trypsin-like enzyme (TrypLE). ( b ) Flow cytometry histograms indicating high purification and survival efficiency of hiPSCs compared to EasySep. ( c ) Flow cytometry scatter plots showing detached UD-hiPSCs (TRA-1-60 + CMPTX + ) and SD-hiPSCs (TRA-1-60 − CMPTX + ) across 10 passages using μSHEAR and TrypLE. ( d ) Enrichment efficiency of hiPSCs when repeatedly passaged by μSHEAR, EDTA, TrypLE, Dispase, or Accutase. hiPSCs from same batch (P 0, 90% TRA-1-60 + ) were used and the recovered culture was propagated for 5–6 days. ( e ) Cell survival on Matrigel after passaging with μSHEAR, manual hand-picking, or TrypLE. ( f ) Growth curves for cells on Matrigel using μSHEAR or hand-picking and starting with an equivalent number of cells at day 0 for each passage (5×10 4 cells). ( g ) Immunostaining for SSEA4 and OCT4 showing μSHEAR-isolated UD-hiPSC cultured on Matrigel retained undifferentiated characteristics across 10 passages. ( h ) Heat-map of expression of stem cell-related and differentiation genes in hiPSCs at P 10 showing no differences in expression between μSHEAR and manual hand-picking, compared to the starting P 0 population. ( i ) Relative expression comparison for stem cell-related genes in isolated hiPSCs at P 10 . Magenta lines indicate two-fold change in gene expression threshold. ( j ) Karyotype analysis of hiPSCs at P 10 . Data report average ± s.d. (* P
Figure Legend Snippet: Adhesive differences in spontaneously differentiated pluripotent stem cells enable adhesive force-based enrichment of undifferentiated cells ( a ) μSHEAR-based isolation of UD-hiPSCs (white arrowhead) from SD-hiPSCs (red arrowhead) at 100 dynes cm −2 with high enrichment efficiency irrespective of SD-hiPSC contamination (table). Bottom panel shows non-selective detachment of cells using a trypsin-like enzyme (TrypLE). ( b ) Flow cytometry histograms indicating high purification and survival efficiency of hiPSCs compared to EasySep. ( c ) Flow cytometry scatter plots showing detached UD-hiPSCs (TRA-1-60 + CMPTX + ) and SD-hiPSCs (TRA-1-60 − CMPTX + ) across 10 passages using μSHEAR and TrypLE. ( d ) Enrichment efficiency of hiPSCs when repeatedly passaged by μSHEAR, EDTA, TrypLE, Dispase, or Accutase. hiPSCs from same batch (P 0, 90% TRA-1-60 + ) were used and the recovered culture was propagated for 5–6 days. ( e ) Cell survival on Matrigel after passaging with μSHEAR, manual hand-picking, or TrypLE. ( f ) Growth curves for cells on Matrigel using μSHEAR or hand-picking and starting with an equivalent number of cells at day 0 for each passage (5×10 4 cells). ( g ) Immunostaining for SSEA4 and OCT4 showing μSHEAR-isolated UD-hiPSC cultured on Matrigel retained undifferentiated characteristics across 10 passages. ( h ) Heat-map of expression of stem cell-related and differentiation genes in hiPSCs at P 10 showing no differences in expression between μSHEAR and manual hand-picking, compared to the starting P 0 population. ( i ) Relative expression comparison for stem cell-related genes in isolated hiPSCs at P 10 . Magenta lines indicate two-fold change in gene expression threshold. ( j ) Karyotype analysis of hiPSCs at P 10 . Data report average ± s.d. (* P

Techniques Used: Isolation, Flow Cytometry, Cytometry, Purification, Passaging, Immunostaining, Cell Culture, Expressing

38) Product Images from "The Absence of CCR7 Results in Dysregulated Monocyte Migration and Immunosuppression Facilitating Chronic Cutaneous Leishmaniasis"

Article Title: The Absence of CCR7 Results in Dysregulated Monocyte Migration and Immunosuppression Facilitating Chronic Cutaneous Leishmaniasis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0079098

CCR7 expression in B6.WT mice at day 14 after infection with L. major . (A) The percentage of CD11c + cells expressing CCR7 was determined by flow cytometry in the footpad, draining lymph node (LN) and spleen. (B) The expression of CCR7 on CD11b + CCR2 + monocytes was determined in each tissue of B6.WT mice. (C) A representative histogram of CCR7 expression on CD11b + CCR2 + monocytes in each tissue is shown. (D) Monocytic populations were defined according to their expression of Ly6C as shown in Figure 2A , and the percentage of cells in each population expressing CCR7 is shown. (E) The percentage of Ly6G + neutrophils in each tissue expressing CCR7 was analysed by flow cytometry. Experimental group size: n=5 mice.
Figure Legend Snippet: CCR7 expression in B6.WT mice at day 14 after infection with L. major . (A) The percentage of CD11c + cells expressing CCR7 was determined by flow cytometry in the footpad, draining lymph node (LN) and spleen. (B) The expression of CCR7 on CD11b + CCR2 + monocytes was determined in each tissue of B6.WT mice. (C) A representative histogram of CCR7 expression on CD11b + CCR2 + monocytes in each tissue is shown. (D) Monocytic populations were defined according to their expression of Ly6C as shown in Figure 2A , and the percentage of cells in each population expressing CCR7 is shown. (E) The percentage of Ly6G + neutrophils in each tissue expressing CCR7 was analysed by flow cytometry. Experimental group size: n=5 mice.

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

39) Product Images from "Immune targeting of fibroblast activation protein triggers recognition of multipotent bone marrow stromal cells and cachexia"

Article Title: Immune targeting of fibroblast activation protein triggers recognition of multipotent bone marrow stromal cells and cachexia

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20130110

Murine and human multipotent BMSCs express FAP and are recognized by T cells expressing FAP-reactive CARs. Passage-5 in vitro–expanded murine BMSCs were stained with antibodies specific for Sca-1, PDGFR-α, and FAP, and assessed by flow cytometry (A). “Q” represents quadrant. Solid lines are isotype controls and filled histograms are FAP stained. UnTd or FAP5-CAR Td T cells were cultured overnight with murine BMSCs and supernatants were assessed for IFN-γ by ELISA (B), and cells were further analyzed for expression of CD107a and production of IFN-γ and TNF by ICS (C). Mean ± SD. Data are representative of two independent experiments. Flow cytometric phenotype of in vitro–expanded human BMSCs derived from three different donors (D). BMSCs from D were stained with the FAP-specific monoclonal antibodies Sibrotuzumab (E) and FAP5 (F) and assessed by flow cytometry. Solid lines are isotype or secondary antibody controls and filled histograms are FAP or Sibrotuzumab stained. UnTd or Sibro-CAR Td T cells were cultured overnight with BMSCs and the supernatants assessed for IFN-γ by ELISA (G), and cells were further analyzed for CD107a expression and IFN-γ and TNF production by ICS (H). Mean ± SD. Similar results were seen with two additional T cell donors.
Figure Legend Snippet: Murine and human multipotent BMSCs express FAP and are recognized by T cells expressing FAP-reactive CARs. Passage-5 in vitro–expanded murine BMSCs were stained with antibodies specific for Sca-1, PDGFR-α, and FAP, and assessed by flow cytometry (A). “Q” represents quadrant. Solid lines are isotype controls and filled histograms are FAP stained. UnTd or FAP5-CAR Td T cells were cultured overnight with murine BMSCs and supernatants were assessed for IFN-γ by ELISA (B), and cells were further analyzed for expression of CD107a and production of IFN-γ and TNF by ICS (C). Mean ± SD. Data are representative of two independent experiments. Flow cytometric phenotype of in vitro–expanded human BMSCs derived from three different donors (D). BMSCs from D were stained with the FAP-specific monoclonal antibodies Sibrotuzumab (E) and FAP5 (F) and assessed by flow cytometry. Solid lines are isotype or secondary antibody controls and filled histograms are FAP or Sibrotuzumab stained. UnTd or Sibro-CAR Td T cells were cultured overnight with BMSCs and the supernatants assessed for IFN-γ by ELISA (G), and cells were further analyzed for CD107a expression and IFN-γ and TNF production by ICS (H). Mean ± SD. Similar results were seen with two additional T cell donors.

Techniques Used: Expressing, In Vitro, Staining, Flow Cytometry, Cytometry, Cell Culture, Enzyme-linked Immunosorbent Assay, Derivative Assay

Expression of FAP on freshly isolated murine BMSCs from OS cells. BM (A) and OS cells (B) were isolated from untreated wild-type C57BL/6 mice and stained with antibodies against CD45, TER119, Sca-1, PDGFR-α, and FAP, followed by flow cytometry analysis. CD45 + /TER119 + cells demarcate hematopoietic and erythroid lineage cells (Lin + ). Expression of FAP in various populations of OS cells stained with antibodies specific for Sca-1 and PDGFR-α (B). Irradiated non–tumor-bearing mice were treated with 2 × 10 7 UnTd or FAP5-CAR Td T cells and, 7 d later, OS cells were isolated and analyzed as in B. Expression of FAP on various OS cell populations isolated from mice treated with UnTd (C) or FAP5-CAR Td (D) T cells is shown. (E) Mean fluorescence intensity (MFI) of FAP in the various Sca-1 and PDGFR-α subsets found in OS cells. All data are gated on live, single cells. Data for C–E are further gated on Lin − (CD45 − /TER119 − ) cells. Solid lines are isotype controls and filled histograms are FAP5 stained. All data are representative of at least two independent experiments.
Figure Legend Snippet: Expression of FAP on freshly isolated murine BMSCs from OS cells. BM (A) and OS cells (B) were isolated from untreated wild-type C57BL/6 mice and stained with antibodies against CD45, TER119, Sca-1, PDGFR-α, and FAP, followed by flow cytometry analysis. CD45 + /TER119 + cells demarcate hematopoietic and erythroid lineage cells (Lin + ). Expression of FAP in various populations of OS cells stained with antibodies specific for Sca-1 and PDGFR-α (B). Irradiated non–tumor-bearing mice were treated with 2 × 10 7 UnTd or FAP5-CAR Td T cells and, 7 d later, OS cells were isolated and analyzed as in B. Expression of FAP on various OS cell populations isolated from mice treated with UnTd (C) or FAP5-CAR Td (D) T cells is shown. (E) Mean fluorescence intensity (MFI) of FAP in the various Sca-1 and PDGFR-α subsets found in OS cells. All data are gated on live, single cells. Data for C–E are further gated on Lin − (CD45 − /TER119 − ) cells. Solid lines are isotype controls and filled histograms are FAP5 stained. All data are representative of at least two independent experiments.

Techniques Used: Expressing, Isolation, Mouse Assay, Staining, Flow Cytometry, Cytometry, Irradiation, Fluorescence

IHC staining for FAP in various human tumors, and design and in vitro activity of FAP-reactive CARs. Representative IHC staining for FAP in human melanoma (A), colorectal (B), pancreatic (C), and breast (D) adenocarcinomas. Isotype stains were negative (not depicted). Bars: 400 µm (A); 200 µm (B–D). Schematic of the FAP-reactive CAR constructs FAP5-CAR (E) and Sibro-CAR (F). LS, GM-CSFR leader sequence; V H and V L , variable heavy and light chains; L, 218 linker; CD8, transmembrane domain; CD28, 4-1BB, and CD3-ζ, intracellular signaling domains; m, murine; h, human. Both constructs were cloned into the MSGV1 retroviral vector. Retrovirus containing FAP5-CAR or Sibro-CAR constructs were generated and used to transduce mouse and human T cells, respectively, and flow cytometry was used to assess transduction efficiency at day 2 after transduction for FAP5-CAR (G) and day 8–10 after transduction for Sibro-CAR (H). Solid line is isotype control and filled histogram is FAP5 or Sibrotuzumab stained. Day 5-stimulated untransduced (UnTd) and FAP5-CAR–transduced (Td) mouse T cells were assessed for reactivity against plate-bound BSA, α-CD3 mAb, and recombinant human FAP (r-huFAP), and against HEK293 cell lines expressing or not expressing FAP. After an overnight stimulation, supernatants were assessed for IFN-γ with an IFN-γ ELISA (I), and cells were further assessed for cell surface CD107a expression, and production of IFN-γ and TNF by ICS (J). For ICS, cells are gated on FAP5-CAR Td cells. Day ∼14-stimulated UnTd or Sibro-CAR Td human T cells were assessed for in vitro reactivity as described for mouse. IFN-γ ELISA (K), and ICS results gated on Sibro-CAR Td T cells (L) are shown. Mean ± SD. All results are representative of at least three independent experiments.
Figure Legend Snippet: IHC staining for FAP in various human tumors, and design and in vitro activity of FAP-reactive CARs. Representative IHC staining for FAP in human melanoma (A), colorectal (B), pancreatic (C), and breast (D) adenocarcinomas. Isotype stains were negative (not depicted). Bars: 400 µm (A); 200 µm (B–D). Schematic of the FAP-reactive CAR constructs FAP5-CAR (E) and Sibro-CAR (F). LS, GM-CSFR leader sequence; V H and V L , variable heavy and light chains; L, 218 linker; CD8, transmembrane domain; CD28, 4-1BB, and CD3-ζ, intracellular signaling domains; m, murine; h, human. Both constructs were cloned into the MSGV1 retroviral vector. Retrovirus containing FAP5-CAR or Sibro-CAR constructs were generated and used to transduce mouse and human T cells, respectively, and flow cytometry was used to assess transduction efficiency at day 2 after transduction for FAP5-CAR (G) and day 8–10 after transduction for Sibro-CAR (H). Solid line is isotype control and filled histogram is FAP5 or Sibrotuzumab stained. Day 5-stimulated untransduced (UnTd) and FAP5-CAR–transduced (Td) mouse T cells were assessed for reactivity against plate-bound BSA, α-CD3 mAb, and recombinant human FAP (r-huFAP), and against HEK293 cell lines expressing or not expressing FAP. After an overnight stimulation, supernatants were assessed for IFN-γ with an IFN-γ ELISA (I), and cells were further assessed for cell surface CD107a expression, and production of IFN-γ and TNF by ICS (J). For ICS, cells are gated on FAP5-CAR Td cells. Day ∼14-stimulated UnTd or Sibro-CAR Td human T cells were assessed for in vitro reactivity as described for mouse. IFN-γ ELISA (K), and ICS results gated on Sibro-CAR Td T cells (L) are shown. Mean ± SD. All results are representative of at least three independent experiments.

Techniques Used: Immunohistochemistry, Staining, In Vitro, Activity Assay, Construct, Sequencing, Clone Assay, Plasmid Preparation, Generated, Transduction, Flow Cytometry, Cytometry, Recombinant, Expressing, Enzyme-linked Immunosorbent Assay

FAP expression in mouse tumors, and in vivo activity of FAP5-CAR–transduced T cells against various murine tumors. In vitro cultured B16, MC38, MC17-51, 4T1, CT26, and Renca murine tumors were assessed for FAP expression by flow cytometry with the FAP-specific antibody FAP5 (A). Solid line is isotype control and filled histogram is FAP5 stained. Results are representative of at least two independent experiments. Established (∼11–16 d) subcutaneously implanted B16 (B), MC38 (C), MC17-51 (D), 4T1 (E), CT26 (F), and Renca (G) tumors were harvested from mice (irradiated before harvest) and assessed for FAP expression by IHC using biotinylated-FAP5 antibody. Bars, 400 µm. Representative of at least two independent experiments. C57BL/6 mice bearing established B16 (H), MC38 (I), MC17-51 (J) tumors, and BALB/c mice bearing established 4T1 (K), CT26 (L), or Renca (M) tumors were left untreated (No Tx) or treated with 10 7 UnTd or 10 7 FAP5-CAR Td T cells, and the perpendicular diameters of the tumors were measured over time. Mean ± SEM. Results are representative of at least two independent experiments for H–J and one experiment for K–M with initially five mice per group. *, P
Figure Legend Snippet: FAP expression in mouse tumors, and in vivo activity of FAP5-CAR–transduced T cells against various murine tumors. In vitro cultured B16, MC38, MC17-51, 4T1, CT26, and Renca murine tumors were assessed for FAP expression by flow cytometry with the FAP-specific antibody FAP5 (A). Solid line is isotype control and filled histogram is FAP5 stained. Results are representative of at least two independent experiments. Established (∼11–16 d) subcutaneously implanted B16 (B), MC38 (C), MC17-51 (D), 4T1 (E), CT26 (F), and Renca (G) tumors were harvested from mice (irradiated before harvest) and assessed for FAP expression by IHC using biotinylated-FAP5 antibody. Bars, 400 µm. Representative of at least two independent experiments. C57BL/6 mice bearing established B16 (H), MC38 (I), MC17-51 (J) tumors, and BALB/c mice bearing established 4T1 (K), CT26 (L), or Renca (M) tumors were left untreated (No Tx) or treated with 10 7 UnTd or 10 7 FAP5-CAR Td T cells, and the perpendicular diameters of the tumors were measured over time. Mean ± SEM. Results are representative of at least two independent experiments for H–J and one experiment for K–M with initially five mice per group. *, P

Techniques Used: Expressing, In Vivo, Activity Assay, In Vitro, Cell Culture, Flow Cytometry, Cytometry, Staining, Mouse Assay, Irradiation, Immunohistochemistry

40) Product Images from "Key Role of Group V Secreted Phospholipase A2 in Th2 Cytokine and Dendritic Cell-Driven Airway Hyperresponsiveness and Remodeling"

Article Title: Key Role of Group V Secreted Phospholipase A2 in Th2 Cytokine and Dendritic Cell-Driven Airway Hyperresponsiveness and Remodeling

Journal: PLoS ONE

doi: 10.1371/journal.pone.0056172

Effect of sPLA 2 -V deficiency on DC OVA uptake, presentation to OVA-transgenic T cells, and PGE 2  production. A.  Endocytosis of Alexa Fluor 488-labeled OVA (0.1 mg/ml) by BMDCs from sPLA 2 -V +/+  (+/+) and sPLA 2 -V −/−  (−/−) mice was assessed at 2 h by flow cytometry using a BD FACSCanto™ Flow Cytometry System with the mean fluorescence intensity (MFI) representing the amount of incorporated tracer by APC-CD11c+ cells ( n  = 4–5, each group).  B.  The antigen-presenting activity of splenic DCs in sPLA2-V-deficient mice in comparison to wild-type controls was assessed using CD4 +  T cells carrying the MHC class II restricted rearranged T cell receptor (TCR) transgene, Tg(DO11.10)10Dlo that react to OVA peptide antigen. Irradiated (3000 rad) splenic DCs from sPLA 2 -V +/+  (+/+) and sPLA 2 -V −/−  (−/−) mice were cultured overnight with OVA (1 mg/ml) at 37°C in 5% CO2; the splenic DCs (0–50000 DCs/well) were then incubated with CD4+ naïve T cells (1×106 T cells/well) isolated from OVA-TCR transgenic mice for 48 h in the absence or presence of OVA323-339 peptide (1 μg/ml).  C.  PGE2 production by BMDCs was assayed by EIA on d 7 in culture after incubation for 24 h with OVA (1 mg/ml) ( n  = 4–5, each group).
Figure Legend Snippet: Effect of sPLA 2 -V deficiency on DC OVA uptake, presentation to OVA-transgenic T cells, and PGE 2 production. A. Endocytosis of Alexa Fluor 488-labeled OVA (0.1 mg/ml) by BMDCs from sPLA 2 -V +/+ (+/+) and sPLA 2 -V −/− (−/−) mice was assessed at 2 h by flow cytometry using a BD FACSCanto™ Flow Cytometry System with the mean fluorescence intensity (MFI) representing the amount of incorporated tracer by APC-CD11c+ cells ( n  = 4–5, each group). B. The antigen-presenting activity of splenic DCs in sPLA2-V-deficient mice in comparison to wild-type controls was assessed using CD4 + T cells carrying the MHC class II restricted rearranged T cell receptor (TCR) transgene, Tg(DO11.10)10Dlo that react to OVA peptide antigen. Irradiated (3000 rad) splenic DCs from sPLA 2 -V +/+ (+/+) and sPLA 2 -V −/− (−/−) mice were cultured overnight with OVA (1 mg/ml) at 37°C in 5% CO2; the splenic DCs (0–50000 DCs/well) were then incubated with CD4+ naïve T cells (1×106 T cells/well) isolated from OVA-TCR transgenic mice for 48 h in the absence or presence of OVA323-339 peptide (1 μg/ml). C. PGE2 production by BMDCs was assayed by EIA on d 7 in culture after incubation for 24 h with OVA (1 mg/ml) ( n  = 4–5, each group).

Techniques Used: Transgenic Assay, Labeling, Mouse Assay, Flow Cytometry, Cytometry, Fluorescence, Activity Assay, Irradiation, Cell Culture, Incubation, Isolation, Enzyme-linked Immunosorbent Assay

Effect of sPLA 2 -V deficiency on CD4 +  T cell and DC proliferation. A.  Cell proliferation of wild-type and sPLA 2 -V −/−  splenic CD4 +  T cells cultured in the absence ( Untreated ) or presence of IL-2 and IL-4 in anti-CD3-coated plates ( IL-2 + IL-4 + anti-CD3 ) for 72 h was determined by MTT assay ( n  = 4–5, each group).  B.  Allogeneic CD4+ cells from C57Bl6 mice were cultured with irradiated (3000 rad) splenic DCs (0–50000 cells/well) from wild-type or sPLA2-V−/− mice for 72 h with cell proliferation measured by MTT assay ( n  = 4–5, each group).  C.  BMDC proliferation was assessed by MTT assay on d 9 after 3 days in culture in the absence ( Untreated ) or presence of GM-CSF and IL-4 ( GM-CSF + IL-4 ) ( n  = 4–5, each group).
Figure Legend Snippet: Effect of sPLA 2 -V deficiency on CD4 + T cell and DC proliferation. A. Cell proliferation of wild-type and sPLA 2 -V −/− splenic CD4 + T cells cultured in the absence ( Untreated ) or presence of IL-2 and IL-4 in anti-CD3-coated plates ( IL-2 + IL-4 + anti-CD3 ) for 72 h was determined by MTT assay ( n  = 4–5, each group). B. Allogeneic CD4+ cells from C57Bl6 mice were cultured with irradiated (3000 rad) splenic DCs (0–50000 cells/well) from wild-type or sPLA2-V−/− mice for 72 h with cell proliferation measured by MTT assay ( n  = 4–5, each group). C. BMDC proliferation was assessed by MTT assay on d 9 after 3 days in culture in the absence ( Untreated ) or presence of GM-CSF and IL-4 ( GM-CSF + IL-4 ) ( n  = 4–5, each group).

Techniques Used: Cell Culture, MTT Assay, Mouse Assay, Irradiation

Single cell immunospot assay of IL-4 and IL-13 production by splenocytes and splenic CD4 +  T cells from sPLA 2 -V −/−  mice. A. Elispot assay of IL-4 and IL-13 production by splenocytes obtained on d 76 from sPLA 2 -V +/+  mice treated with saline (+/+  Saline ) or OVA (+/+  OVA ) and sPLA 2 -V −/−  mice treated with saline ( − / − Saline ) or OVA (−/−  OVA ) and incubated in the absence ( Untreated ) or presence of either 500 μg/ml OVA ( OVA ) or 5 ng/ml PMA/500 ng/ml ionomycin ( PMA + Ionomycin ) ( n  = 4-5, each group).  B.  Elispot assay of IL-4 and IL-13 production by CD4 +  T cells isolated from the total splenic cells obtained on d 76 from sPLA 2 -V +/+  mice incubated in the absence ( Untreated ) or presence of anti-CD3/anti-CD28 antibodies ( anti-CD3/anti-CD28 ).
Figure Legend Snippet: Single cell immunospot assay of IL-4 and IL-13 production by splenocytes and splenic CD4 + T cells from sPLA 2 -V −/− mice. A. Elispot assay of IL-4 and IL-13 production by splenocytes obtained on d 76 from sPLA 2 -V +/+ mice treated with saline (+/+ Saline ) or OVA (+/+ OVA ) and sPLA 2 -V −/− mice treated with saline ( − / − Saline ) or OVA (−/− OVA ) and incubated in the absence ( Untreated ) or presence of either 500 μg/ml OVA ( OVA ) or 5 ng/ml PMA/500 ng/ml ionomycin ( PMA + Ionomycin ) ( n  = 4-5, each group). B. Elispot assay of IL-4 and IL-13 production by CD4 + T cells isolated from the total splenic cells obtained on d 76 from sPLA 2 -V +/+ mice incubated in the absence ( Untreated ) or presence of anti-CD3/anti-CD28 antibodies ( anti-CD3/anti-CD28 ).

Techniques Used: Mouse Assay, Enzyme-linked Immunospot, Incubation, Isolation

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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).

Staining:

Article Title: Co-stimulatory function in primary germinal center responses: CD40 and B7 are required on distinct antigen-presenting cells
Article Snippet: .. For intracellular cytokine staining, splenic CD4+ T cells were stimulated with PMA and ionomycin for 2 h, and cells were fixed and permeabilized with the BD Fix/Perm kit (BD Biosciences) according to the manufacturer’s instructions and then stained with anti–IFN-γ (BD Biosciences) and isotype control antibody for 30 min. Data were collected with a FACS Calibur II, FACS LSR II, FACS Fortessa, or FACS Aria III flow cytometer (BD Biosciences) and analyzed with FlowJo software. .. ELISA NP-specific IgG1 was measured by ELISA.

Article Title: Lenalidomide regulates CNS autoimmunity by promoting M2 macrophages polarization
Article Snippet: .. Single-cell suspensions from DLN, spleen, and CNS were incubated for 30 min at 4 °C with fluorochrome-conjugated anti-CD4, anti-CD8, anti-CD45, anti-B220 (all purchased from BD Biosciences), anti-F4/80, anti-CD11b, anti-CD86, and anti-CD206 (all purchased from Biolegend) for staining of surface markers. .. For intracellular staining of cytokines, the cells were stained with anti-CD4, followed by staining with anti-IFN-γ, anti-IL17A (all purchased from eBioScience) and anti-IL10 (Biolegend) using Cytofix/Cytoperm kit (BD Biosciences) according to the manufacturer’s protocol.

Article Title: TIGIT and PD-1 dual checkpoint blockade enhances antitumor immunity and survival in GBM
Article Snippet: .. Immune cells were stained for surface markers using fluorescence-conjugated monoclonal antibodies including CD45 (clone HI30, BD Biosciences), CD3 (clone UCHT1, BD Biosciences), CD4 (clone L200, BD Biosciences), CD8 (clone SK1, BD Biosciences), PD1 (clone EH12.1, BD Biosciences), and TIGIT (clone 741182, R & D Systems). .. Flow cytometry was performed using FACS LSR Fortessa™ (BD Biosciences), and data was analyzed using FlowJo software (Treestar).

Article Title: Antigen-selective modulation of AAV immunogenicity with tolerogenic rapamycin nanoparticles enables successful vector re-administration
Article Snippet: .. Flow cytometry analysis Single-cell suspensions from spleen and lymph nodes were prepared and stained for the surface markers with different fluorochrome combinations: anti-CD3 (APC-eFluor780, clone 17A2, dilution 1:300, BD Biosciences, San Jose, CA), anti-CD4 (PB, clone RM4-5, dilution 1:200, BD Biosciences), anti-CD25 (PE, clone PC61, dilution 1:300 BD Biosciences), anti-CD45R (FITC, clone RA3.6B2, dilution 1:300, Thermo Fisher Scientific Waltham, MA), anti-CD19 (PE, clone 1D3, dilution 1:300 BD Biosciences), anti-IgD (APC-eFluor780, clone 11-26 c.2a, dilution 1:200, BD Biosciences), anti-GL7 (BV421, clone GL7, dilution 1:100, BD Biosciences), anti-CD95 (PE-Cy7, clone Jo2, dilution 1:200, BD Biosciences), and anti-PD− 1 (BV605, clone J43, dilution 1:200, BD Biosciences), followed by cell viability staining using Fixable Live/Dead kit (Biolegend, San Diego, CA) according to manufacturer’s instructions. .. For the detection of mouse CXCR5, cells were first stained with biotin conjugated anti-CXCR5 or with biotin conjugated with isotype control (clone SPRCL5, dilution 1:50, Thermo Fisher Scientific) followed by streptavidin-PE (dilution 1:200, Thermo Fisher Scientific).

FACS:

Article Title: Co-stimulatory function in primary germinal center responses: CD40 and B7 are required on distinct antigen-presenting cells
Article Snippet: .. For intracellular cytokine staining, splenic CD4+ T cells were stimulated with PMA and ionomycin for 2 h, and cells were fixed and permeabilized with the BD Fix/Perm kit (BD Biosciences) according to the manufacturer’s instructions and then stained with anti–IFN-γ (BD Biosciences) and isotype control antibody for 30 min. Data were collected with a FACS Calibur II, FACS LSR II, FACS Fortessa, or FACS Aria III flow cytometer (BD Biosciences) and analyzed with FlowJo software. .. ELISA NP-specific IgG1 was measured by ELISA.

Software:

Article Title: Co-stimulatory function in primary germinal center responses: CD40 and B7 are required on distinct antigen-presenting cells
Article Snippet: .. For intracellular cytokine staining, splenic CD4+ T cells were stimulated with PMA and ionomycin for 2 h, and cells were fixed and permeabilized with the BD Fix/Perm kit (BD Biosciences) according to the manufacturer’s instructions and then stained with anti–IFN-γ (BD Biosciences) and isotype control antibody for 30 min. Data were collected with a FACS Calibur II, FACS LSR II, FACS Fortessa, or FACS Aria III flow cytometer (BD Biosciences) and analyzed with FlowJo software. .. ELISA NP-specific IgG1 was measured by ELISA.

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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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    94
    Becton Dickinson anti cd107a
    Influence of FcRn on NK cell functions and in vitro expansion. Purified splenic NK cells were analyzed by flow cytometry for (A) the intracellular measurement of IFN-γ and (B) the surface expression of the late endosomal marker <t>CD107a,</t> after 4-h incubation at 37°C without (Ctr) or with PMA (100 ng/mL)/ionomycine (500 ng/mL). Data are median ± Min to Max analyzed from eight independent experiments using pooled NK cells from 2 mice. Freshly isolated splenocytes were seeded in RMPI 1640 complete medium supplemented with 5,000 U/ml rhIL2 or with 5 ng/ml rhIL12, 50 ng/ml rhIL15, and 10 ng/ml rhIL18 for 4-h (C,D) . Within splenocytes, CD3 − /NK1.1 + /NKp46 + cells were analyzed for (C) the intracellular expression of IFN-γ and (D) the surface expression of CD107a by flow cytometry. Data are median ± Min to Max from two independent experiments using pooled spleens from 2 mice. (E) Cytotoxicity assay was performed against CFSE-labeled YAC-1 target cells with different ratios of purified NK cells previously maintained overnight in RPMI 1640 complete medium supplemented with 50 U/ml of rhIL2 ( n = 3). The results were expressed as means ± SEM. (F,G) Purified splenic NK cells were plated in complete medium supplemented with 5,000 U/ml rhIL2. (F) The living cell numbers and (G) the percentage of dead cells were determined daily by manual cell counting using trypan blue in Malassez chamber ( n = 3). The results were expressed as mean ± SEM (F) and median ± Min to Max (G) . ns = not significant * p ≤ 0.05 and ** p ≤ 0.005 using two-tailed non-parametric and unpaired Wilcoxon-Mann-Whitney test.
    Anti Cd107a, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 174 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Becton Dickinson flow cytometry antibody characterization
    hASCs demonstrate pericyte-like morphology and phenotype markers in vitro and in vivo . A B–C , DiI-labeled hASCs (red) wrap around isolectin labeled retinal microvessels (blue) abluminally and target vascular junctions, both properties of terminally differentiated pericytes. D–I , Passage 5 hASCs exhibit in virto expression of the characteristic pericyte markers smooth muscle actin (SMA, D–E ), nerve/glial antigen 2 (NG2, F–G ), and platelet derived growth factor receptor beta (PDGFR-β, H–I ) by both immumohistochemical staining on cultured cells and by flow <t>cytometry</t> on cells harvested from these cultures. J , 91.2% of hASCs demonstrated colabeling of SMA and PDGFR-β compared to unstained controls. K–L , DiI labeled hASCs injected intravitreally into NOD SCID mice at P12, after OIR hyperoxia, maintain intimate association with the retinal microvasculature 6–8 weeks later and demonstrate persistent SMA ( K ), NG2 ( L ) (white arrows), with SMA extending into the cellular extension wrapping around the capillary. On average 308 hASCs, or 3.08% of the 10,000 injected cells, remained engrafted, with 85.6% of engrafted hASCs found in physical contact with retinal microvessels. Note native retinal pericytes are also labeled with NG2 ( L ) but lack DiI staining (white arrowheads). In contrast, no native retinal pericytes are seen labeled with SMA ( K ) given that this is a tertiary branch of the retinal vasculature, and native pericyte SMA expression is typically seen only on primary and secondary vessels. Scale bars: A = 200 µm, B = 20 µm, C = 10 µm, D, F, H = 100 µm, K = 10 µm, L = 20 µm.
    Flow Cytometry Antibody Characterization, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Becton Dickinson anti mouse igm
    Emv2 -selected CD4 + T cells retain full antiviral activity. (A) Mean frequency (± SEM, n = 8–19) of FV-infected (glyco-Gag + ) Ter119 + cells in the spleens of FV-infected B6 or Emv2 -deficient B6 mice (B6- Emv2 −/− ). (B–C) CD4 + T cells isolated from either B6 (B6-EF4.1) or Emv2 -deficient B6 (B6-EF4.1 Emv2 −/− ) EF4.1 mice were adoptively transferred into B6 or B6.A- Fv2 s recipients that were infected with FV the same day and analyzed 7 days later. (B) Flow cytometric example of FV-infected Ter119 + cells from B6 recipients and (C) frequency of FV-infected cells in Ter119 + cells from the spleens of B6 or B6.A- Fv2 s recipients of CD4 + T cells. Control B6 and B6.A- Fv2 s mice that received no CD4 + T cells (-) are also included. Each symbol is an individual mouse. (D) Spleen index ( left ) and RBC count ( right ) of B6- Rag1 −/− Fv2 s mice that were infected with FV and either received the same day CD4 + T cells isolated from either B6 (B6-EF4.1) or Emv2 -deficient B6 (B6-EF4.1 Emv2 −/− ) EF4.1 mice or no cells (-). Each symbol is an individual mouse analyzed 3 weeks post infection. (E) Titers of FV-neutralizing antibodies during the course of FV infection ( left ) and titers of F-MLV-infected cell-binding IgG ( middle ) and <t>IgM</t> ( right ) 7 days post FV infection, in the sera of B6- Tcra −/− mice that either received CD4 + T cells isolated from either B6 (B6-EF4.1) or Emv2 -deficient B6 (B6-EF4.1 Emv2 −/− ) EF4.1 mice or no cells (-) the day of the infection. Dashed lines represent the limit of detection. Data are the means ± SEM ( n = 11–12) from 2 experiments.
    Anti Mouse Igm, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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

    Influence of FcRn on NK cell functions and in vitro expansion. Purified splenic NK cells were analyzed by flow cytometry for (A) the intracellular measurement of IFN-γ and (B) the surface expression of the late endosomal marker CD107a, after 4-h incubation at 37°C without (Ctr) or with PMA (100 ng/mL)/ionomycine (500 ng/mL). Data are median ± Min to Max analyzed from eight independent experiments using pooled NK cells from 2 mice. Freshly isolated splenocytes were seeded in RMPI 1640 complete medium supplemented with 5,000 U/ml rhIL2 or with 5 ng/ml rhIL12, 50 ng/ml rhIL15, and 10 ng/ml rhIL18 for 4-h (C,D) . Within splenocytes, CD3 − /NK1.1 + /NKp46 + cells were analyzed for (C) the intracellular expression of IFN-γ and (D) the surface expression of CD107a by flow cytometry. Data are median ± Min to Max from two independent experiments using pooled spleens from 2 mice. (E) Cytotoxicity assay was performed against CFSE-labeled YAC-1 target cells with different ratios of purified NK cells previously maintained overnight in RPMI 1640 complete medium supplemented with 50 U/ml of rhIL2 ( n = 3). The results were expressed as means ± SEM. (F,G) Purified splenic NK cells were plated in complete medium supplemented with 5,000 U/ml rhIL2. (F) The living cell numbers and (G) the percentage of dead cells were determined daily by manual cell counting using trypan blue in Malassez chamber ( n = 3). The results were expressed as mean ± SEM (F) and median ± Min to Max (G) . ns = not significant * p ≤ 0.05 and ** p ≤ 0.005 using two-tailed non-parametric and unpaired Wilcoxon-Mann-Whitney test.

    Journal: Frontiers in Immunology

    Article Title: Lack of FcRn Impairs Natural Killer Cell Development and Functions in the Tumor Microenvironment

    doi: 10.3389/fimmu.2018.02259

    Figure Lengend Snippet: Influence of FcRn on NK cell functions and in vitro expansion. Purified splenic NK cells were analyzed by flow cytometry for (A) the intracellular measurement of IFN-γ and (B) the surface expression of the late endosomal marker CD107a, after 4-h incubation at 37°C without (Ctr) or with PMA (100 ng/mL)/ionomycine (500 ng/mL). Data are median ± Min to Max analyzed from eight independent experiments using pooled NK cells from 2 mice. Freshly isolated splenocytes were seeded in RMPI 1640 complete medium supplemented with 5,000 U/ml rhIL2 or with 5 ng/ml rhIL12, 50 ng/ml rhIL15, and 10 ng/ml rhIL18 for 4-h (C,D) . Within splenocytes, CD3 − /NK1.1 + /NKp46 + cells were analyzed for (C) the intracellular expression of IFN-γ and (D) the surface expression of CD107a by flow cytometry. Data are median ± Min to Max from two independent experiments using pooled spleens from 2 mice. (E) Cytotoxicity assay was performed against CFSE-labeled YAC-1 target cells with different ratios of purified NK cells previously maintained overnight in RPMI 1640 complete medium supplemented with 50 U/ml of rhIL2 ( n = 3). The results were expressed as means ± SEM. (F,G) Purified splenic NK cells were plated in complete medium supplemented with 5,000 U/ml rhIL2. (F) The living cell numbers and (G) the percentage of dead cells were determined daily by manual cell counting using trypan blue in Malassez chamber ( n = 3). The results were expressed as mean ± SEM (F) and median ± Min to Max (G) . ns = not significant * p ≤ 0.05 and ** p ≤ 0.005 using two-tailed non-parametric and unpaired Wilcoxon-Mann-Whitney test.

    Article Snippet: Next, 105 freshly isolated NK cells were added per well with 5 μl (0.5 mg/ml) anti-CD107a (clone 1D4B, FITC, Becton Dickinson [BD]) and 1 μg/106 cells of brefeldin A (GolgiPlug, BD).

    Techniques: In Vitro, Purification, Flow Cytometry, Cytometry, Expressing, Marker, Incubation, Mouse Assay, Isolation, Cytotoxicity Assay, Labeling, Cell Counting, Two Tailed Test, MANN-WHITNEY

    hASCs demonstrate pericyte-like morphology and phenotype markers in vitro and in vivo . A B–C , DiI-labeled hASCs (red) wrap around isolectin labeled retinal microvessels (blue) abluminally and target vascular junctions, both properties of terminally differentiated pericytes. D–I , Passage 5 hASCs exhibit in virto expression of the characteristic pericyte markers smooth muscle actin (SMA, D–E ), nerve/glial antigen 2 (NG2, F–G ), and platelet derived growth factor receptor beta (PDGFR-β, H–I ) by both immumohistochemical staining on cultured cells and by flow cytometry on cells harvested from these cultures. J , 91.2% of hASCs demonstrated colabeling of SMA and PDGFR-β compared to unstained controls. K–L , DiI labeled hASCs injected intravitreally into NOD SCID mice at P12, after OIR hyperoxia, maintain intimate association with the retinal microvasculature 6–8 weeks later and demonstrate persistent SMA ( K ), NG2 ( L ) (white arrows), with SMA extending into the cellular extension wrapping around the capillary. On average 308 hASCs, or 3.08% of the 10,000 injected cells, remained engrafted, with 85.6% of engrafted hASCs found in physical contact with retinal microvessels. Note native retinal pericytes are also labeled with NG2 ( L ) but lack DiI staining (white arrowheads). In contrast, no native retinal pericytes are seen labeled with SMA ( K ) given that this is a tertiary branch of the retinal vasculature, and native pericyte SMA expression is typically seen only on primary and secondary vessels. Scale bars: A = 200 µm, B = 20 µm, C = 10 µm, D, F, H = 100 µm, K = 10 µm, L = 20 µm.

    Journal: PLoS ONE

    Article Title: Pericytes Derived from Adipose-Derived Stem Cells Protect against Retinal Vasculopathy

    doi: 10.1371/journal.pone.0065691

    Figure Lengend Snippet: hASCs demonstrate pericyte-like morphology and phenotype markers in vitro and in vivo . A B–C , DiI-labeled hASCs (red) wrap around isolectin labeled retinal microvessels (blue) abluminally and target vascular junctions, both properties of terminally differentiated pericytes. D–I , Passage 5 hASCs exhibit in virto expression of the characteristic pericyte markers smooth muscle actin (SMA, D–E ), nerve/glial antigen 2 (NG2, F–G ), and platelet derived growth factor receptor beta (PDGFR-β, H–I ) by both immumohistochemical staining on cultured cells and by flow cytometry on cells harvested from these cultures. J , 91.2% of hASCs demonstrated colabeling of SMA and PDGFR-β compared to unstained controls. K–L , DiI labeled hASCs injected intravitreally into NOD SCID mice at P12, after OIR hyperoxia, maintain intimate association with the retinal microvasculature 6–8 weeks later and demonstrate persistent SMA ( K ), NG2 ( L ) (white arrows), with SMA extending into the cellular extension wrapping around the capillary. On average 308 hASCs, or 3.08% of the 10,000 injected cells, remained engrafted, with 85.6% of engrafted hASCs found in physical contact with retinal microvessels. Note native retinal pericytes are also labeled with NG2 ( L ) but lack DiI staining (white arrowheads). In contrast, no native retinal pericytes are seen labeled with SMA ( K ) given that this is a tertiary branch of the retinal vasculature, and native pericyte SMA expression is typically seen only on primary and secondary vessels. Scale bars: A = 200 µm, B = 20 µm, C = 10 µm, D, F, H = 100 µm, K = 10 µm, L = 20 µm.

    Article Snippet: Flow Cytometry Antibody characterization of hASCs (passage 4) was performed on a Becton Dickinson/Cytek FACSCalibur C with CellQuest Pro acquisition software, using FlowJo software for analysis.

    Techniques: In Vitro, In Vivo, Labeling, Expressing, Derivative Assay, Staining, Cell Culture, Flow Cytometry, Cytometry, Injection, Mouse Assay

    Emv2 -selected CD4 + T cells retain full antiviral activity. (A) Mean frequency (± SEM, n = 8–19) of FV-infected (glyco-Gag + ) Ter119 + cells in the spleens of FV-infected B6 or Emv2 -deficient B6 mice (B6- Emv2 −/− ). (B–C) CD4 + T cells isolated from either B6 (B6-EF4.1) or Emv2 -deficient B6 (B6-EF4.1 Emv2 −/− ) EF4.1 mice were adoptively transferred into B6 or B6.A- Fv2 s recipients that were infected with FV the same day and analyzed 7 days later. (B) Flow cytometric example of FV-infected Ter119 + cells from B6 recipients and (C) frequency of FV-infected cells in Ter119 + cells from the spleens of B6 or B6.A- Fv2 s recipients of CD4 + T cells. Control B6 and B6.A- Fv2 s mice that received no CD4 + T cells (-) are also included. Each symbol is an individual mouse. (D) Spleen index ( left ) and RBC count ( right ) of B6- Rag1 −/− Fv2 s mice that were infected with FV and either received the same day CD4 + T cells isolated from either B6 (B6-EF4.1) or Emv2 -deficient B6 (B6-EF4.1 Emv2 −/− ) EF4.1 mice or no cells (-). Each symbol is an individual mouse analyzed 3 weeks post infection. (E) Titers of FV-neutralizing antibodies during the course of FV infection ( left ) and titers of F-MLV-infected cell-binding IgG ( middle ) and IgM ( right ) 7 days post FV infection, in the sera of B6- Tcra −/− mice that either received CD4 + T cells isolated from either B6 (B6-EF4.1) or Emv2 -deficient B6 (B6-EF4.1 Emv2 −/− ) EF4.1 mice or no cells (-) the day of the infection. Dashed lines represent the limit of detection. Data are the means ± SEM ( n = 11–12) from 2 experiments.

    Journal: PLoS Pathogens

    Article Title: Negative Selection by an Endogenous Retrovirus Promotes a Higher-Avidity CD4+ T Cell Response to Retroviral Infection

    doi: 10.1371/journal.ppat.1002709

    Figure Lengend Snippet: Emv2 -selected CD4 + T cells retain full antiviral activity. (A) Mean frequency (± SEM, n = 8–19) of FV-infected (glyco-Gag + ) Ter119 + cells in the spleens of FV-infected B6 or Emv2 -deficient B6 mice (B6- Emv2 −/− ). (B–C) CD4 + T cells isolated from either B6 (B6-EF4.1) or Emv2 -deficient B6 (B6-EF4.1 Emv2 −/− ) EF4.1 mice were adoptively transferred into B6 or B6.A- Fv2 s recipients that were infected with FV the same day and analyzed 7 days later. (B) Flow cytometric example of FV-infected Ter119 + cells from B6 recipients and (C) frequency of FV-infected cells in Ter119 + cells from the spleens of B6 or B6.A- Fv2 s recipients of CD4 + T cells. Control B6 and B6.A- Fv2 s mice that received no CD4 + T cells (-) are also included. Each symbol is an individual mouse. (D) Spleen index ( left ) and RBC count ( right ) of B6- Rag1 −/− Fv2 s mice that were infected with FV and either received the same day CD4 + T cells isolated from either B6 (B6-EF4.1) or Emv2 -deficient B6 (B6-EF4.1 Emv2 −/− ) EF4.1 mice or no cells (-). Each symbol is an individual mouse analyzed 3 weeks post infection. (E) Titers of FV-neutralizing antibodies during the course of FV infection ( left ) and titers of F-MLV-infected cell-binding IgG ( middle ) and IgM ( right ) 7 days post FV infection, in the sera of B6- Tcra −/− mice that either received CD4 + T cells isolated from either B6 (B6-EF4.1) or Emv2 -deficient B6 (B6-EF4.1 Emv2 −/− ) EF4.1 mice or no cells (-) the day of the infection. Dashed lines represent the limit of detection. Data are the means ± SEM ( n = 11–12) from 2 experiments.

    Article Snippet: Serum titers of F-MLV-infected cell-binding antibodies were determined by flow cytometry following primary staining of F-MLV-infected Mus dunni cells with serial dilutions of serum samples and secondary staining with fluorescently labeled anti-mouse IgG1 (clone A85-1), anti-mouse IgG2a/c (clone R19-15), anti-mouse IgG2b (clone R12-3) or anti-mouse IgM (clone R6-60.2) antibodies (BD).

    Techniques: Activity Assay, Infection, Mouse Assay, Isolation, Flow Cytometry, Binding Assay