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 "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

3) 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

4) 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

5) 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

6) 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

7) 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

8) 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

9) Product Images from "Combined use of anti-ErbB monoclonal antibodies and erlotinib enhances antibody-dependent cellular cytotoxicity of wild-type erlotinib-sensitive NSCLC cell lines"

Article Title: Combined use of anti-ErbB monoclonal antibodies and erlotinib enhances antibody-dependent cellular cytotoxicity of wild-type erlotinib-sensitive NSCLC cell lines

Journal: Molecular Cancer

doi: 10.1186/1476-4598-11-91

EGFR and HER2 increase at the plasma-membrane level. Calu-3 (A) and H292 (B) cell lines were treated with 1 μM erlotinib for 24 h, H322 cell line was treated with increasing concentration of erlotinib (C) or with 1 μM erlotinib for the indicated period of time (D) . At the end of the treatment, cell surface expression of EGFR and/or HER2 were evaluated by flow cytometry and the quantification is reported as Molecules of Equivalent Fluorophore [MEF] or as fold increase versus untreated control cells (D) . Inset Figure 2A : Western blot analysis of EGFR protein membrane level in Calu-3 after treatment with 1 μM erlotinib for 24 h. Whole cells were labeled with biotin and membrane bound proteins were pulled down with neutrAvidin beads. The results are from representative experiments. Each experiment, repeated three times, yielded similar results.
Figure Legend Snippet: EGFR and HER2 increase at the plasma-membrane level. Calu-3 (A) and H292 (B) cell lines were treated with 1 μM erlotinib for 24 h, H322 cell line was treated with increasing concentration of erlotinib (C) or with 1 μM erlotinib for the indicated period of time (D) . At the end of the treatment, cell surface expression of EGFR and/or HER2 were evaluated by flow cytometry and the quantification is reported as Molecules of Equivalent Fluorophore [MEF] or as fold increase versus untreated control cells (D) . Inset Figure 2A : Western blot analysis of EGFR protein membrane level in Calu-3 after treatment with 1 μM erlotinib for 24 h. Whole cells were labeled with biotin and membrane bound proteins were pulled down with neutrAvidin beads. The results are from representative experiments. Each experiment, repeated three times, yielded similar results.

Techniques Used: Concentration Assay, Expressing, Flow Cytometry, Cytometry, Western Blot, Labeling

Erlotinib induces EGFR and HER2 protein accumulation only in sensitive NSCLC cell lines. (A) Calu-3, H322, H292, PC9 and HCC827 cell lines were treated with the indicated concentrations of erlotinib for 48 h. At the end of the drug treatment cell lysates were immunoblotted to detect the indicated proteins. The immunoreactive spots were quantified by densitometric analysis, ratios of EGFR/Actin and HER2/Actin were calculated at 1 μM erlotinib for Calu-3 H322 and H292 or 10 nM for PC9 and HCC827 and values are expressed as fold increase versus control (B) . (C) HCC827GR5, A549, H1299, H1703, Calu-1 cell lines were treated with 1 μM erlotinib for 48 h and at the end of treatment cell lysates were immunoblotted to detect the indicated proteins. The immunoreactive spots were quantified by densitometric analysis, ratios of EGFR/Actin and HER2/Actin were calculated and values are expressed as fold increase versus control. (D) Representative Western blotting of resistant H1299 cell line exposed to increased concentration of erlotinib. (E) HCC827 parental cell line and HCC827GR5 resistant clone were treated with the indicated doses of gefitinib and processed as above. The results are from representative experiments. Each experiment, repeated three times, yielded similar results.
Figure Legend Snippet: Erlotinib induces EGFR and HER2 protein accumulation only in sensitive NSCLC cell lines. (A) Calu-3, H322, H292, PC9 and HCC827 cell lines were treated with the indicated concentrations of erlotinib for 48 h. At the end of the drug treatment cell lysates were immunoblotted to detect the indicated proteins. The immunoreactive spots were quantified by densitometric analysis, ratios of EGFR/Actin and HER2/Actin were calculated at 1 μM erlotinib for Calu-3 H322 and H292 or 10 nM for PC9 and HCC827 and values are expressed as fold increase versus control (B) . (C) HCC827GR5, A549, H1299, H1703, Calu-1 cell lines were treated with 1 μM erlotinib for 48 h and at the end of treatment cell lysates were immunoblotted to detect the indicated proteins. The immunoreactive spots were quantified by densitometric analysis, ratios of EGFR/Actin and HER2/Actin were calculated and values are expressed as fold increase versus control. (D) Representative Western blotting of resistant H1299 cell line exposed to increased concentration of erlotinib. (E) HCC827 parental cell line and HCC827GR5 resistant clone were treated with the indicated doses of gefitinib and processed as above. The results are from representative experiments. Each experiment, repeated three times, yielded similar results.

Techniques Used: Western Blot, Concentration Assay

10) Product Images from "A Higher Frequency of Circulating IL-22+CD4+ T Cells in Chinese Patients with Newly Diagnosed Hashimoto's Thyroiditis"

Article Title: A Higher Frequency of Circulating IL-22+CD4+ T Cells in Chinese Patients with Newly Diagnosed Hashimoto's Thyroiditis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0084545

Flow cytometry analysis of the frequency of different subsets of CD4+ T cells. PBMCs were isolated from individual participants and stimulated with PMA and ionomycin for six hours in the presence of BFA. The cells were stained with fluorescent antibodies against CD3 and CD4, fixed and permeabilized, followed by intracellular staining with anti-IFN-γ, anti-IL-17A and anti-IL-22. The cells were gated on CD3+CD4+ T cells and the percentages of IFN-γ − IL-17A − IL-22 + , IFN-γ − IL-22 − IL-17A + IFN-γ − IL-17A + IL-22 + , IFN-γ + IL-17A − IL-22 − and IFN-γ + CD4 + T cells in CD3+CD4+ T cells were determined. Data are representative dot plots and the percentages of different subsets of CD4+ T cells in the HT patients and HC. The horizontal lines indicate the median values.
Figure Legend Snippet: Flow cytometry analysis of the frequency of different subsets of CD4+ T cells. PBMCs were isolated from individual participants and stimulated with PMA and ionomycin for six hours in the presence of BFA. The cells were stained with fluorescent antibodies against CD3 and CD4, fixed and permeabilized, followed by intracellular staining with anti-IFN-γ, anti-IL-17A and anti-IL-22. The cells were gated on CD3+CD4+ T cells and the percentages of IFN-γ − IL-17A − IL-22 + , IFN-γ − IL-22 − IL-17A + IFN-γ − IL-17A + IL-22 + , IFN-γ + IL-17A − IL-22 − and IFN-γ + CD4 + T cells in CD3+CD4+ T cells were determined. Data are representative dot plots and the percentages of different subsets of CD4+ T cells in the HT patients and HC. The horizontal lines indicate the median values.

Techniques Used: Flow Cytometry, Cytometry, Isolation, Staining

11) Product Images from "A One Year Follow-Up Study of Natural Killer and Dendritic Cells Activities in Multiple Sclerosis Patients Receiving Glatiramer Acetate (GA)"

Article Title: A One Year Follow-Up Study of Natural Killer and Dendritic Cells Activities in Multiple Sclerosis Patients Receiving Glatiramer Acetate (GA)

Journal: PLoS ONE

doi: 10.1371/journal.pone.0062237

Treatment with GA changes expression of surface molecules in iDCs from RRMS patients. The expression of HLA-DR, HLA-I, HLA-E as well as the co-stimulatory molecules CD80, CD83 or CD86 was analyzed by flow cytometry of iDCs from RRMS patients before (pre) and weeks after treatment-start. Mean fluorescence intensity (MFI) of 10000 analyzed cells is shown.
Figure Legend Snippet: Treatment with GA changes expression of surface molecules in iDCs from RRMS patients. The expression of HLA-DR, HLA-I, HLA-E as well as the co-stimulatory molecules CD80, CD83 or CD86 was analyzed by flow cytometry of iDCs from RRMS patients before (pre) and weeks after treatment-start. Mean fluorescence intensity (MFI) of 10000 analyzed cells is shown.

Techniques Used: Expressing, Flow Cytometry, Cytometry, Fluorescence

Treatment with GA changes expression of surface molecules in mDCs from RRMS patients. The expression of HLA-DR, HLA-I, HLA-E as well as co-stimulatory molecules CD80, CD83 or CD86 was analyzed with flow cytometry of mDCs from RRMS patient before (Pre) and weeks after treatment start. Mean fluorescence intensity (MFI) of 10000 analyzed cells is shown.
Figure Legend Snippet: Treatment with GA changes expression of surface molecules in mDCs from RRMS patients. The expression of HLA-DR, HLA-I, HLA-E as well as co-stimulatory molecules CD80, CD83 or CD86 was analyzed with flow cytometry of mDCs from RRMS patient before (Pre) and weeks after treatment start. Mean fluorescence intensity (MFI) of 10000 analyzed cells is shown.

Techniques Used: Expressing, Flow Cytometry, Cytometry, Fluorescence

12) Product Images from "CD11b−CD27− NK Cells Are Associated with the Progression of Lung Carcinoma"

Article Title: CD11b−CD27− NK Cells Are Associated with the Progression of Lung Carcinoma

Journal: PLoS ONE

doi: 10.1371/journal.pone.0061024

The DN NK subset in TINK cells accounts for the immature phenotype. (A) Representative flow cytometry analysis of the expression of various surface molecules (CD16, CD57, CD127, CD117, CD226 and NKp30) on the DN NK subset versus the CD11b + SP NK subset in TINK cells. Dot plots were gated on live NK cells using a lymphocyte gate based on forward scatter versus side scatter and an NK-cell gate identifying CD56 + CD3 − cells. DN NK cells were identified based on gating for CD56 + CD3 − CD11b − CD27 − , while CD11b + SP NK cells were identified based on a CD56 + CD3 − CD11b + CD27 − gate. Quadrants depicted were set on isotype controls. (B) The major phenotypic differences detected in the two subsets (DN and CD11b + SP NK) are summarised. Data shown represent the findings from 15 patients (mean±SD).
Figure Legend Snippet: The DN NK subset in TINK cells accounts for the immature phenotype. (A) Representative flow cytometry analysis of the expression of various surface molecules (CD16, CD57, CD127, CD117, CD226 and NKp30) on the DN NK subset versus the CD11b + SP NK subset in TINK cells. Dot plots were gated on live NK cells using a lymphocyte gate based on forward scatter versus side scatter and an NK-cell gate identifying CD56 + CD3 − cells. DN NK cells were identified based on gating for CD56 + CD3 − CD11b − CD27 − , while CD11b + SP NK cells were identified based on a CD56 + CD3 − CD11b + CD27 − gate. Quadrants depicted were set on isotype controls. (B) The major phenotypic differences detected in the two subsets (DN and CD11b + SP NK) are summarised. Data shown represent the findings from 15 patients (mean±SD).

Techniques Used: Flow Cytometry, Cytometry, Expressing

TINK cells display an inactive phenotype. (A) Representative flow cytometry analysis of the expression of NK-cell activation receptors (CD16, CD226 and NKp30) on gated CD56 + CD3 − TINK cells as compared with that on pNK cells from autologous patients and healthy control subjects. Quadrants depicted were set on isotype controls. (B) The frequency of CD16 + , CD226 + and NKp30 + NK cells within the above-mentioned three NK-cell populations (n = 15; mean±SEM).
Figure Legend Snippet: TINK cells display an inactive phenotype. (A) Representative flow cytometry analysis of the expression of NK-cell activation receptors (CD16, CD226 and NKp30) on gated CD56 + CD3 − TINK cells as compared with that on pNK cells from autologous patients and healthy control subjects. Quadrants depicted were set on isotype controls. (B) The frequency of CD16 + , CD226 + and NKp30 + NK cells within the above-mentioned three NK-cell populations (n = 15; mean±SEM).

Techniques Used: Flow Cytometry, Cytometry, Expressing, Activation Assay

13) Product Images from "Mapping the Homodimer Interface of an Optimized, Artificial, Transmembrane Protein Activator of the Human Erythropoietin Receptor"

Article Title: Mapping the Homodimer Interface of an Optimized, Artificial, Transmembrane Protein Activator of the Human Erythropoietin Receptor

Journal: PLoS ONE

doi: 10.1371/journal.pone.0095593

EBC5-16 displays increased ability to stimulate erythroid differentiation of human hematopoietic progenitor cells. (A) Primary human CD34 + cells infected with retrovirus expressing empty CMMP-IRES-GFP vector (green), or CMMP-IRES-GFP expressing TC2-3 (red) or EBC5-16 (blue) were sorted for GFP fluorescence and transferred to differentiation medium in the absence of EPO. A sample of cells expressing vector was also treated with EPO (magenta). After six days in differentiation medium, viable cells were assessed for cell-surface GpA expression by immunostaining and flow cytometry. Similar results were obtained in four independent experiments. (B) Cells were handled as in (A). After six days in differentiation medium, the total number of viable cells expressing GpA ( > 50 fluorescence units) was determined by immunostaining and flow cytometry. Graph shows average of three independent experiments. Error bars represent the standard error of the mean. A student t-test determined the difference between EBC5-16 and TC2-3 samples to be statistically significant, p
Figure Legend Snippet: EBC5-16 displays increased ability to stimulate erythroid differentiation of human hematopoietic progenitor cells. (A) Primary human CD34 + cells infected with retrovirus expressing empty CMMP-IRES-GFP vector (green), or CMMP-IRES-GFP expressing TC2-3 (red) or EBC5-16 (blue) were sorted for GFP fluorescence and transferred to differentiation medium in the absence of EPO. A sample of cells expressing vector was also treated with EPO (magenta). After six days in differentiation medium, viable cells were assessed for cell-surface GpA expression by immunostaining and flow cytometry. Similar results were obtained in four independent experiments. (B) Cells were handled as in (A). After six days in differentiation medium, the total number of viable cells expressing GpA ( > 50 fluorescence units) was determined by immunostaining and flow cytometry. Graph shows average of three independent experiments. Error bars represent the standard error of the mean. A student t-test determined the difference between EBC5-16 and TC2-3 samples to be statistically significant, p

Techniques Used: Infection, Expressing, Plasmid Preparation, Fluorescence, Immunostaining, Flow Cytometry, Cytometry

14) Product Images from "A Novel Copper Chelate Modulates Tumor Associated Macrophages to Promote Anti-Tumor Response of T Cells"

Article Title: A Novel Copper Chelate Modulates Tumor Associated Macrophages to Promote Anti-Tumor Response of T Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0007048

In vitro CuNG treatment caused reprogramming of Treg. A, B C) Flow cytometry. CD4 + CD25 + Treg populations were purified from TALs of untreated EAC/Dox bearing mice. (A) Treg cells were labeled with CFSE and then cultured for 96 h with cell free supernatant of TAMs (isolated from untreated EAC/Dox bearing mice) either kept untreated or treated in vitro with CuNG for 48 h. Intracellular IFN-γ and TGF-β production was analyzed with respect to specific isotype control by flow cytometry. Representative data of three independent experiments is shown. (B) Treg cells were cultured for 96 h with supernatant of 48 h culture of untreated or in vitro CuNG treated TAMs and fresh medium (1∶1). Intracellular IFN-γ and TGF-β production versus FoxP3 expression was analyzed with respect to specific isotype control by flow cytometry. Representative data of four independent experiments is shown. (C) Tregs (CD4 + CD25 + cells) isolated from ascitic fluid of untreated EAC/Dox bearing mice were cultured for 96 h in presence of cell-free supernatants from 48 h cultures of untreated or CuNG treated TAMs (culture supernatant: fresh medium being 1∶1). Now, these cells were washed and cultured with CFSE loaded CD4 + T cells isolated from inguinal and axillary lymph nodes of normal mice (Treg and CD4 + T cells were taken in a proportion of 1∶5) for 96 h. Fluorescence levels of CFSE were measured by flow cytometry. Proliferation of normal CD4 + T cells either in the presence or absence of Treg cells were also analyzed by CFSE fluorescence level. Representative data of 3 independent experiments is presented here.
Figure Legend Snippet: In vitro CuNG treatment caused reprogramming of Treg. A, B C) Flow cytometry. CD4 + CD25 + Treg populations were purified from TALs of untreated EAC/Dox bearing mice. (A) Treg cells were labeled with CFSE and then cultured for 96 h with cell free supernatant of TAMs (isolated from untreated EAC/Dox bearing mice) either kept untreated or treated in vitro with CuNG for 48 h. Intracellular IFN-γ and TGF-β production was analyzed with respect to specific isotype control by flow cytometry. Representative data of three independent experiments is shown. (B) Treg cells were cultured for 96 h with supernatant of 48 h culture of untreated or in vitro CuNG treated TAMs and fresh medium (1∶1). Intracellular IFN-γ and TGF-β production versus FoxP3 expression was analyzed with respect to specific isotype control by flow cytometry. Representative data of four independent experiments is shown. (C) Tregs (CD4 + CD25 + cells) isolated from ascitic fluid of untreated EAC/Dox bearing mice were cultured for 96 h in presence of cell-free supernatants from 48 h cultures of untreated or CuNG treated TAMs (culture supernatant: fresh medium being 1∶1). Now, these cells were washed and cultured with CFSE loaded CD4 + T cells isolated from inguinal and axillary lymph nodes of normal mice (Treg and CD4 + T cells were taken in a proportion of 1∶5) for 96 h. Fluorescence levels of CFSE were measured by flow cytometry. Proliferation of normal CD4 + T cells either in the presence or absence of Treg cells were also analyzed by CFSE fluorescence level. Representative data of 3 independent experiments is presented here.

Techniques Used: In Vitro, Flow Cytometry, Cytometry, Purification, Mouse Assay, Labeling, Cell Culture, Isolation, Expressing, Fluorescence

Both in vitro and in vivo CuNG treatment caused alteration of cytokines profile of TAMs. A) ELISA. B) Flow cytometry. C) Fluorometric analysis. D, E F) ELISA. In vitro CuNG treatment (2.5 µg/ml) did not change IFN-γ production from CD4 + T cells of TALs of untreated EAC/Dox bearing mice (A). TAMs were purified from peritoneal ascitic fluid of both untreated and 15 days of CuNG treated EAC/Dox bearing mice and labeled with anti F4/80 antibodies and with either intracellular IL-10 or IL-12 or TGF-β or with specific isotype control Abs. Immunofluorescence analysis were performed by flow cytometry. Representative data of 3 independent experiments is presented (B). Purified TAMs were either kept untreated or treated with CuNG in vitro and ROS was measured [in terms of peroxide using dichlorofluorescein diacetate (DCF-DA)] at different time points. Results are presented as mean±SD of 3 independent experiments (C). Purified TAMs from untreated EAC/Dox bearing mice were plated (2×10 6 cells/500 µl). Cells were either kept untreated or pretreated with tocopherol (50 µM) for 1 h. Then the cells were further cultured for 12 h, 24 h and 48 h in the presence or absence of CuNG (2.5 µg/ml). The culture supernatants were collected and analyzed for cytokines IL-10 (D), IL-12 (E) and TGF-β (F) by ELISA and results are presented as mean±SE of 3 independent experiments, each experiment having every measurement in triplicate.
Figure Legend Snippet: Both in vitro and in vivo CuNG treatment caused alteration of cytokines profile of TAMs. A) ELISA. B) Flow cytometry. C) Fluorometric analysis. D, E F) ELISA. In vitro CuNG treatment (2.5 µg/ml) did not change IFN-γ production from CD4 + T cells of TALs of untreated EAC/Dox bearing mice (A). TAMs were purified from peritoneal ascitic fluid of both untreated and 15 days of CuNG treated EAC/Dox bearing mice and labeled with anti F4/80 antibodies and with either intracellular IL-10 or IL-12 or TGF-β or with specific isotype control Abs. Immunofluorescence analysis were performed by flow cytometry. Representative data of 3 independent experiments is presented (B). Purified TAMs were either kept untreated or treated with CuNG in vitro and ROS was measured [in terms of peroxide using dichlorofluorescein diacetate (DCF-DA)] at different time points. Results are presented as mean±SD of 3 independent experiments (C). Purified TAMs from untreated EAC/Dox bearing mice were plated (2×10 6 cells/500 µl). Cells were either kept untreated or pretreated with tocopherol (50 µM) for 1 h. Then the cells were further cultured for 12 h, 24 h and 48 h in the presence or absence of CuNG (2.5 µg/ml). The culture supernatants were collected and analyzed for cytokines IL-10 (D), IL-12 (E) and TGF-β (F) by ELISA and results are presented as mean±SE of 3 independent experiments, each experiment having every measurement in triplicate.

Techniques Used: In Vitro, In Vivo, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cytometry, Mouse Assay, Purification, Labeling, Immunofluorescence, Cell Culture

Combination of high IL-12 and low IL-10 can skew induction of Th1 response. A) RT-PCR. B) Densitometric analysis. C, D, E F) Flow cytometry. CD4 + population from TALs (obtained from untreated EAC/Dox bearing mice) was purified and challenged either with single or combine dose of recombinant IL-12 and IL-10 and cultured for 96 h. Purified CD4 + population from TALs derived from untreated EAC/Dox bearing mice, cultured without any treatment was taken as untreated control. Equivalent amount of mRNA (2 µg) from each experimental group was used for semi-quantitative RT-PCR analysis and in all cases GAPDH was used as housekeeping gene control (A). Densitometry analysis of mRNA expression of each gene transcript was expressed as a ratio of cytokine mRNA to GAPDH mRNA (B). Intracellular cytokines specific for Th1 (IFN-γ) or Th2 (IL-4) or suppressive (TGF-β) production profile in the above mentioned experimental groups were also analyzed by flow cytometry and a representative data is shown (C). CD4+ TALs were co-cultured with untreated TAMs or CuNG treated TAMs either unfixed or fixed with paraformaldehyde or CuNG treated fixed TAMs along with high rIL-12 and low rIL-10. In some cases CD4+ TALs and CuNG treated unfixed TAMs were separated by transwell insert (0.45 µ Meter pore) in culture. After 96 h of culture intracellular IFN-γ and TGF-β production pattern were studied by flow cytometry (D). Mean fluorescence intensity for IFN-γ ( Fig. 5E ) and TGF-β ( Fig. 5F ) production by these experimental groups were also analyzed from the flow cytometric statistical data and represented graphically. Representative data from three independent experiments is presented.
Figure Legend Snippet: Combination of high IL-12 and low IL-10 can skew induction of Th1 response. A) RT-PCR. B) Densitometric analysis. C, D, E F) Flow cytometry. CD4 + population from TALs (obtained from untreated EAC/Dox bearing mice) was purified and challenged either with single or combine dose of recombinant IL-12 and IL-10 and cultured for 96 h. Purified CD4 + population from TALs derived from untreated EAC/Dox bearing mice, cultured without any treatment was taken as untreated control. Equivalent amount of mRNA (2 µg) from each experimental group was used for semi-quantitative RT-PCR analysis and in all cases GAPDH was used as housekeeping gene control (A). Densitometry analysis of mRNA expression of each gene transcript was expressed as a ratio of cytokine mRNA to GAPDH mRNA (B). Intracellular cytokines specific for Th1 (IFN-γ) or Th2 (IL-4) or suppressive (TGF-β) production profile in the above mentioned experimental groups were also analyzed by flow cytometry and a representative data is shown (C). CD4+ TALs were co-cultured with untreated TAMs or CuNG treated TAMs either unfixed or fixed with paraformaldehyde or CuNG treated fixed TAMs along with high rIL-12 and low rIL-10. In some cases CD4+ TALs and CuNG treated unfixed TAMs were separated by transwell insert (0.45 µ Meter pore) in culture. After 96 h of culture intracellular IFN-γ and TGF-β production pattern were studied by flow cytometry (D). Mean fluorescence intensity for IFN-γ ( Fig. 5E ) and TGF-β ( Fig. 5F ) production by these experimental groups were also analyzed from the flow cytometric statistical data and represented graphically. Representative data from three independent experiments is presented.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Flow Cytometry, Cytometry, Mouse Assay, Purification, Recombinant, Cell Culture, Derivative Assay, Quantitative RT-PCR, Expressing, Fluorescence

Presence of small amount of IL-10 prolonged Th1 response by delaying T cells apoptosis. A) Flow cytometry. B) Fluorometric analysis. C D) Flow cytometry. Purified CD4 + population from TALs of untreated EAC/Dox bearing mice cultured in the presence of either only rIL-12 or the combination of high rIL-12 (2.7 ng/ml) and low rIL-10 (0.35 ng/ml) or the culture supernatant of in vitro CuNG treated (48 h of CuNG treatment) TAMs or IL-10 neutralized culture supernatant of in vitro CuNG treated (48 h of CuNG treatment) TAMs or 48 h of culture supernatant from in vivo CuNG treated TAMs or IL-10 neutralized culture supernatant of in vivo CuNG treated TAMs, for 72 h, 96 h and 120 h. Purified CD4 + population without any treatment was taken as untreated control. Levels of apoptosis were estimated by PI/Annexin V-FITC staining and flow cytometry. Representative data of 3 independent experiments is presented here (A). Purified CD4 + population pre-treated with H2O2 for 30 mins was taken as positive control for active caspase 3 level (Mean fluorescence intensity value of H2O2 control was 39.25±0.67 that was taken as 100% for active caspase 3 level). In each experimental group active caspase 3 levels was represented by % of H 2 O 2 positive control. Results presented are of 4 independent experiments (B). Expression of Fas by CD4 + population of above mentioned experimental groups were also analyzed by flow cytometry. Cells were labeled with Abs specific for CD4 and for surface Fas or with specific isotype Abs and immunofluorescence analysis was performed. Representative result of 4 independent experiments is presented (C). CD4 + TALs were cultured with or without different combinations of rIL-12 and rIL-10 in absence or presence of neutralizing antibody against IFN-γ for 72 h. Fas expression was studied by flow cytometry and representative data of 3 independent experiments is presented here (D).
Figure Legend Snippet: Presence of small amount of IL-10 prolonged Th1 response by delaying T cells apoptosis. A) Flow cytometry. B) Fluorometric analysis. C D) Flow cytometry. Purified CD4 + population from TALs of untreated EAC/Dox bearing mice cultured in the presence of either only rIL-12 or the combination of high rIL-12 (2.7 ng/ml) and low rIL-10 (0.35 ng/ml) or the culture supernatant of in vitro CuNG treated (48 h of CuNG treatment) TAMs or IL-10 neutralized culture supernatant of in vitro CuNG treated (48 h of CuNG treatment) TAMs or 48 h of culture supernatant from in vivo CuNG treated TAMs or IL-10 neutralized culture supernatant of in vivo CuNG treated TAMs, for 72 h, 96 h and 120 h. Purified CD4 + population without any treatment was taken as untreated control. Levels of apoptosis were estimated by PI/Annexin V-FITC staining and flow cytometry. Representative data of 3 independent experiments is presented here (A). Purified CD4 + population pre-treated with H2O2 for 30 mins was taken as positive control for active caspase 3 level (Mean fluorescence intensity value of H2O2 control was 39.25±0.67 that was taken as 100% for active caspase 3 level). In each experimental group active caspase 3 levels was represented by % of H 2 O 2 positive control. Results presented are of 4 independent experiments (B). Expression of Fas by CD4 + population of above mentioned experimental groups were also analyzed by flow cytometry. Cells were labeled with Abs specific for CD4 and for surface Fas or with specific isotype Abs and immunofluorescence analysis was performed. Representative result of 4 independent experiments is presented (C). CD4 + TALs were cultured with or without different combinations of rIL-12 and rIL-10 in absence or presence of neutralizing antibody against IFN-γ for 72 h. Fas expression was studied by flow cytometry and representative data of 3 independent experiments is presented here (D).

Techniques Used: Flow Cytometry, Cytometry, Purification, Mouse Assay, Cell Culture, In Vitro, In Vivo, Staining, Positive Control, Fluorescence, Expressing, Labeling, Immunofluorescence

TALs of EAC/Dox bearing mice showed Th1 specific response after in vivo administration of CuNG. Flow cytometry. EAC/Dox bearing mice (n = 12) were treated with CuNG (5 mg/kg of body weight), i.m., 7 days following inoculation and 15 days after CuNG administration TALs were isolated from the ascitic fluid of treated and untreated animals as nonadherent population (method described in material and method section). From isolated TALs, CD4 vs. intracellular IFN-γ, IL-4 and TGF-β production were analyzed by flow cytometry and significantly higher percentage of CD4 population of treated group showed positive for IFN-γ (marker for Th1 response). A representative result is presented here for comparison.
Figure Legend Snippet: TALs of EAC/Dox bearing mice showed Th1 specific response after in vivo administration of CuNG. Flow cytometry. EAC/Dox bearing mice (n = 12) were treated with CuNG (5 mg/kg of body weight), i.m., 7 days following inoculation and 15 days after CuNG administration TALs were isolated from the ascitic fluid of treated and untreated animals as nonadherent population (method described in material and method section). From isolated TALs, CD4 vs. intracellular IFN-γ, IL-4 and TGF-β production were analyzed by flow cytometry and significantly higher percentage of CD4 population of treated group showed positive for IFN-γ (marker for Th1 response). A representative result is presented here for comparison.

Techniques Used: Mouse Assay, In Vivo, Flow Cytometry, Cytometry, Isolation, Marker

15) Product Images from "CD8+ T Cells as a Source of IFN-? Production in Human Cutaneous Leishmaniasis"

Article Title: CD8+ T Cells as a Source of IFN-? Production in Human Cutaneous Leishmaniasis

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0000845

Cytokine profile of CD4 + and CD8 + T cells after SLA stimulation. Purified CD4 + and CD8 + T cells were adjusted to 1–2×10 5 cells/well in a U-bottomed 96-well plates and co-cultured with 1∶10 of mitomycin treated autologous monocytes in cRPMI 1640 supplemented with 10% human AB Rh+ serum. IFN-γ, IL-5, IL-10, and IL-13 were titrated on supernatant of SLA stimulated of CD4 + (A) and CD8 + (B) T cells at 72 hrs of culture using sandwich ELISA method. The amount of IL-5 was not detectable in the culture supernatants. Filled symbols represent HCL volunteers; Open symbols represent healthy controls.
Figure Legend Snippet: Cytokine profile of CD4 + and CD8 + T cells after SLA stimulation. Purified CD4 + and CD8 + T cells were adjusted to 1–2×10 5 cells/well in a U-bottomed 96-well plates and co-cultured with 1∶10 of mitomycin treated autologous monocytes in cRPMI 1640 supplemented with 10% human AB Rh+ serum. IFN-γ, IL-5, IL-10, and IL-13 were titrated on supernatant of SLA stimulated of CD4 + (A) and CD8 + (B) T cells at 72 hrs of culture using sandwich ELISA method. The amount of IL-5 was not detectable in the culture supernatants. Filled symbols represent HCL volunteers; Open symbols represent healthy controls.

Techniques Used: Purification, Cell Culture, Sandwich ELISA

Frequency of purified CD4 + /CD8 + T cells producing intracellular IFN-γ. A portion of the cells at 72 hrs of SLA stimulation was used for ICS assay. Cells were adjusted at about 5×10 5 /ml and stimulated with PMA + Ionomycin for 5–6 hrs. Monensin was added during the last 4–5 hrs of culture. Cells were permeabilized and stained for intracellular IFN-γ with conjugated mAbs. A) One representative flow cytometry plot showing intracellular IFN-γ positive fractions of gated populations of CD4 + and CD8 + T cells in HCL volunteers. B) One representative flow cytometry plot showing intracellular IFN-γ positive fractions of gated populations of CD4 + and CD8 + T cells in healthy controls. C) Flow cytometry data of all volunteers were pooled and are shown as Median (horizontal line) with interquartile ranges (box) and range (whiskers) of intracellular IFN-γ positive cells.
Figure Legend Snippet: Frequency of purified CD4 + /CD8 + T cells producing intracellular IFN-γ. A portion of the cells at 72 hrs of SLA stimulation was used for ICS assay. Cells were adjusted at about 5×10 5 /ml and stimulated with PMA + Ionomycin for 5–6 hrs. Monensin was added during the last 4–5 hrs of culture. Cells were permeabilized and stained for intracellular IFN-γ with conjugated mAbs. A) One representative flow cytometry plot showing intracellular IFN-γ positive fractions of gated populations of CD4 + and CD8 + T cells in HCL volunteers. B) One representative flow cytometry plot showing intracellular IFN-γ positive fractions of gated populations of CD4 + and CD8 + T cells in healthy controls. C) Flow cytometry data of all volunteers were pooled and are shown as Median (horizontal line) with interquartile ranges (box) and range (whiskers) of intracellular IFN-γ positive cells.

Techniques Used: Purification, Staining, Flow Cytometry, Cytometry

16) Product Images from "Rapid mobilization of murine and human hematopoietic stem and progenitor cells with AMD3100, a CXCR4 antagonist"

Article Title: Rapid mobilization of murine and human hematopoietic stem and progenitor cells with AMD3100, a CXCR4 antagonist

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20041385

Influence of AMD3100, G-CSF, and the combination of G-CSF plus AMD3100 on mobilization of NOD-SCID SRCs from normal human volunteers (apheresis samples), surface expression of adhesion molecules and chemotaxis of CD34 + cells, and homing of mobilized murine Sca1 + Lin − cells. (A) SRCs per kg in apheresis samples from G-CSF– ( n = 3), G-CSF + AMD3100- (160 μg/kg; n = 3), and AMD3100- (240 μg/kg; n = 4) mobilized circulating blood. Each set of test samples was assayed simultaneously in limiting dilutions in conditioned NOD-SCID mice. For every sample, four different cell concentrations were used and four mice were transplanted with each cell concentration. Mice were assayed for chimerism 8 wk later; those that demonstrated > 0.2% chimerism (total CD45 + cells in BM) were considered to be positive. Percentage of negative mice were used to calculate SRC frequencies. (B, i) Expression of CD49d (VLA-4), CD49e (VLA-5), CD26L (L-selectin), and CXCR4 on mobilized CD34 + cells (mean ± 1SEM of percent positive cells of five to seven different G-CSF samples, three AMD3100 plus G-CSF samples, and four BM samples). (B, ii) Mean fluorescent intensity (MFI) of positive samples. Same number of samples evaluated as in B, i except G-CSF group has a different number ( n =7). (C) CD34 + cells isolated from G-CSF ( n = 6) and G-CSF plus AMD3100 ( n = 3) mobilized peripheral blood were assessed for chemotaxis to SDF-1/CXCL12 (100 ng/ml) and results expressed as percentage of migrated cells. Data for each sample were collected in duplicate. (D, i) Homing of CD45.2 + Sca1 + Lin − BM cells from C57Bl/6 mice into lethally irradiated CD45.1 + B6.BoyJ BM from samples mobilized with G-CSF (2 times/d for 4 d as in Fig. 2B ; n = 3), G-CSF + AMD3100 (5 mg/kg; n = 4), and AMD3100 (5 mg/kg; n = 3). Homing was assessed as in Materials and methods. (D, ii) Competitive repopulation of CD45.2 + BM cells recovered after homing shown in D, i. Results of chimerism are after 4 mo in 2° irradiated B6.BoyJ recipients. For A–D, significant differences compared with G-CSF: *P
Figure Legend Snippet: Influence of AMD3100, G-CSF, and the combination of G-CSF plus AMD3100 on mobilization of NOD-SCID SRCs from normal human volunteers (apheresis samples), surface expression of adhesion molecules and chemotaxis of CD34 + cells, and homing of mobilized murine Sca1 + Lin − cells. (A) SRCs per kg in apheresis samples from G-CSF– ( n = 3), G-CSF + AMD3100- (160 μg/kg; n = 3), and AMD3100- (240 μg/kg; n = 4) mobilized circulating blood. Each set of test samples was assayed simultaneously in limiting dilutions in conditioned NOD-SCID mice. For every sample, four different cell concentrations were used and four mice were transplanted with each cell concentration. Mice were assayed for chimerism 8 wk later; those that demonstrated > 0.2% chimerism (total CD45 + cells in BM) were considered to be positive. Percentage of negative mice were used to calculate SRC frequencies. (B, i) Expression of CD49d (VLA-4), CD49e (VLA-5), CD26L (L-selectin), and CXCR4 on mobilized CD34 + cells (mean ± 1SEM of percent positive cells of five to seven different G-CSF samples, three AMD3100 plus G-CSF samples, and four BM samples). (B, ii) Mean fluorescent intensity (MFI) of positive samples. Same number of samples evaluated as in B, i except G-CSF group has a different number ( n =7). (C) CD34 + cells isolated from G-CSF ( n = 6) and G-CSF plus AMD3100 ( n = 3) mobilized peripheral blood were assessed for chemotaxis to SDF-1/CXCL12 (100 ng/ml) and results expressed as percentage of migrated cells. Data for each sample were collected in duplicate. (D, i) Homing of CD45.2 + Sca1 + Lin − BM cells from C57Bl/6 mice into lethally irradiated CD45.1 + B6.BoyJ BM from samples mobilized with G-CSF (2 times/d for 4 d as in Fig. 2B ; n = 3), G-CSF + AMD3100 (5 mg/kg; n = 4), and AMD3100 (5 mg/kg; n = 3). Homing was assessed as in Materials and methods. (D, ii) Competitive repopulation of CD45.2 + BM cells recovered after homing shown in D, i. Results of chimerism are after 4 mo in 2° irradiated B6.BoyJ recipients. For A–D, significant differences compared with G-CSF: *P

Techniques Used: Expressing, Chemotaxis Assay, Mouse Assay, Concentration Assay, Isolation, Irradiation

AMD3100 mobilizes competitive repopulating mouse HSCs with self-renewal capacity (A) and G-CSF synergizes this effect (B). (A, left) 18 C57Bl/6 mice (CD45.2) serving as donors were injected s.c. with 0.1 ml saline, and 36 C57Bl/6 mice were injected with 0.1 ml AMD3100 at 5 mg/kg (=∼100 μg/mouse). Blood was collected 1 h later and LDMNCs isolated. BM from nonirradiated B6.BoyJ (CD45.1) mice served as competitor cells. The ratio of donor (CD45.2) blood cells to competitor (CD45.1) BM cells was set as the number of LDMNC blood cells in 3 donor mice to a constant number of 0.5 × 10 6 competitor BM cells equaling 3:1. A 2:1 ratio was the number of LDMNCs in blood of 2 donor mice to 0.5 × 10 6 competitor cells, and a 1:1 ratio was LDMNC in 1 donor mouse to 0.5 × 10 6 competitor cells. Results are based on analysis of transplantation of 6 lethally irradiated recipients per test group of i.v. infused cells. Right panel: BM cells were removed from femurs of primary mice 4 mo after injection of 3:1 saline or 3:1 AMD3100:competitor cell mixture and 2.5 × 10 6 marrow cells from 3 mice of each group injected separately into 3 lethally secondary irradiated B6.BoyJ mice in a noncompetitive assay. Results are shown as percent CD45.2 C57Bl/6 donor cell chimerism in CD45.1 B6.BoyJ recipients. *P
Figure Legend Snippet: AMD3100 mobilizes competitive repopulating mouse HSCs with self-renewal capacity (A) and G-CSF synergizes this effect (B). (A, left) 18 C57Bl/6 mice (CD45.2) serving as donors were injected s.c. with 0.1 ml saline, and 36 C57Bl/6 mice were injected with 0.1 ml AMD3100 at 5 mg/kg (=∼100 μg/mouse). Blood was collected 1 h later and LDMNCs isolated. BM from nonirradiated B6.BoyJ (CD45.1) mice served as competitor cells. The ratio of donor (CD45.2) blood cells to competitor (CD45.1) BM cells was set as the number of LDMNC blood cells in 3 donor mice to a constant number of 0.5 × 10 6 competitor BM cells equaling 3:1. A 2:1 ratio was the number of LDMNCs in blood of 2 donor mice to 0.5 × 10 6 competitor cells, and a 1:1 ratio was LDMNC in 1 donor mouse to 0.5 × 10 6 competitor cells. Results are based on analysis of transplantation of 6 lethally irradiated recipients per test group of i.v. infused cells. Right panel: BM cells were removed from femurs of primary mice 4 mo after injection of 3:1 saline or 3:1 AMD3100:competitor cell mixture and 2.5 × 10 6 marrow cells from 3 mice of each group injected separately into 3 lethally secondary irradiated B6.BoyJ mice in a noncompetitive assay. Results are shown as percent CD45.2 C57Bl/6 donor cell chimerism in CD45.1 B6.BoyJ recipients. *P

Techniques Used: Mouse Assay, Injection, Isolation, Transplantation Assay, Irradiation

17) Product Images from "Leukotriene B4, an activation product of mast cells, is a chemoattractant for their progenitors"

Article Title: Leukotriene B4, an activation product of mast cells, is a chemoattractant for their progenitors

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20042407

Characterization of BMMC cultures. The percentage of c-kit + cells in the total BMMC culture (a) and the mean fluorescence intensity of c-kit expression on the c-kit + BMMCs (b) were measured by flow cytometry. Data shown are ±SEM ( n = 9). (c) Representative dot plots showing double staining of 2-wk- (left), 6-wk- (middle), and 10-wk-old (right) BMMCs with anti–c-kit–PE (c-kit) and either IgE followed by anti-IgE–FITC (top, FcɛR1), anti-CD34–FITC (top middle, CD34), anti-T1/ST2–FITC (bottom middle, T1/ST2), or anti-CD13–FITC (bottom, CD13). Data shown are ±SEM ( n = 6). (d) The mean fluorescence intensity of α 4 and β 7 integrins on c-kit + BMMCs decreases from 1 to 10 wk. (e) Representative dot plots showing forward and side scatter of 2-wk- (left), 6-wk- (middle), and 10-wk-old (right) BMMCs. Data shown are ±SEM ( n = 4).
Figure Legend Snippet: Characterization of BMMC cultures. The percentage of c-kit + cells in the total BMMC culture (a) and the mean fluorescence intensity of c-kit expression on the c-kit + BMMCs (b) were measured by flow cytometry. Data shown are ±SEM ( n = 9). (c) Representative dot plots showing double staining of 2-wk- (left), 6-wk- (middle), and 10-wk-old (right) BMMCs with anti–c-kit–PE (c-kit) and either IgE followed by anti-IgE–FITC (top, FcɛR1), anti-CD34–FITC (top middle, CD34), anti-T1/ST2–FITC (bottom middle, T1/ST2), or anti-CD13–FITC (bottom, CD13). Data shown are ±SEM ( n = 6). (d) The mean fluorescence intensity of α 4 and β 7 integrins on c-kit + BMMCs decreases from 1 to 10 wk. (e) Representative dot plots showing forward and side scatter of 2-wk- (left), 6-wk- (middle), and 10-wk-old (right) BMMCs. Data shown are ±SEM ( n = 4).

Techniques Used: Fluorescence, Expressing, Flow Cytometry, Cytometry, Double Staining

18) Product Images from "Circulating microparticles carry oxidation-specific epitopes and are recognized by natural IgM antibodies 1Circulating microparticles carry oxidation-specific epitopes and are recognized by natural IgM antibodies 1 [S]"

Article Title: Circulating microparticles carry oxidation-specific epitopes and are recognized by natural IgM antibodies 1Circulating microparticles carry oxidation-specific epitopes and are recognized by natural IgM antibodies 1 [S]

Journal: Journal of Lipid Research

doi: 10.1194/jlr.P054569

A subset of circulating MPs carry OSEs. A, B: MPs isolated from plasma of healthy volunteers were stained with annexin V and OSE-specific IgM NAbs including T15/E06 (specific for PC), LR04, NA17, and E014 (specific for MDA-type epitopes) and analyzed by flow cytometry. A: Representative flow cytometry plots of stained MPs. B: Quantification of the percentages of MPs with positive staining for each antibody. Bonferroni’s multiple comparison test; $ P
Figure Legend Snippet: A subset of circulating MPs carry OSEs. A, B: MPs isolated from plasma of healthy volunteers were stained with annexin V and OSE-specific IgM NAbs including T15/E06 (specific for PC), LR04, NA17, and E014 (specific for MDA-type epitopes) and analyzed by flow cytometry. A: Representative flow cytometry plots of stained MPs. B: Quantification of the percentages of MPs with positive staining for each antibody. Bonferroni’s multiple comparison test; $ P

Techniques Used: Isolation, Staining, Multiple Displacement Amplification, Flow Cytometry, Cytometry

LR04, an MDA-specific IgM NAb, decreases the proinflammatory effect of platelet MPs. A: Platelet-derived MPs stained with the MDA/MAA-specific LR04 or isotype antibody and analyzed by flow cytometry. B: Stimulation of THP-1 human monocytes for 8 h with in vitro generated platelet-derived MPs resulted in IL-8 secretion, which was inhibited when MPs were preincubated with LR04 compared with isotype control. Data are from one experiment representative of four in triplicate determinations. C: Stimulation of primary human monocytes isolated from healthy donors (n = 6) for 8 h with in vitro generated platelet-derived MPs resulted in IL-8 secretion, which was inhibited when MPs were preincubated with LR04 but not an isotype control. Data are from two independent experiments. Data are presented as mean ± SEM (ns, not significant; ** P
Figure Legend Snippet: LR04, an MDA-specific IgM NAb, decreases the proinflammatory effect of platelet MPs. A: Platelet-derived MPs stained with the MDA/MAA-specific LR04 or isotype antibody and analyzed by flow cytometry. B: Stimulation of THP-1 human monocytes for 8 h with in vitro generated platelet-derived MPs resulted in IL-8 secretion, which was inhibited when MPs were preincubated with LR04 compared with isotype control. Data are from one experiment representative of four in triplicate determinations. C: Stimulation of primary human monocytes isolated from healthy donors (n = 6) for 8 h with in vitro generated platelet-derived MPs resulted in IL-8 secretion, which was inhibited when MPs were preincubated with LR04 but not an isotype control. Data are from two independent experiments. Data are presented as mean ± SEM (ns, not significant; ** P

Techniques Used: Multiple Displacement Amplification, Derivative Assay, Staining, Flow Cytometry, Cytometry, In Vitro, Generated, Isolation

Circulating MPs carry IgMs that have specificity for MDA-LDL. A: IgM antibodies are bound on the surface of circulating MPs. MPs isolated from healthy volunteers were stained with anti-human IgM and an isotype control antibody and analyzed by flow cytometry. Symbols depict percentages of MPs with bound IgM of individual donors. B: IgM antibodies eluted from circulating MPs are enriched for IgM with specificity for MDA-LDL compared with IgM in plasma. Binding of MP-eluted and plasma IgM to native LDL, Cu-OxLDL, and MDA-LDL was measured by ELISA, and the ratio of antigen-specific IgM/total IgM was calculated. Shown are the mean ± SEM results of four samples. *** P
Figure Legend Snippet: Circulating MPs carry IgMs that have specificity for MDA-LDL. A: IgM antibodies are bound on the surface of circulating MPs. MPs isolated from healthy volunteers were stained with anti-human IgM and an isotype control antibody and analyzed by flow cytometry. Symbols depict percentages of MPs with bound IgM of individual donors. B: IgM antibodies eluted from circulating MPs are enriched for IgM with specificity for MDA-LDL compared with IgM in plasma. Binding of MP-eluted and plasma IgM to native LDL, Cu-OxLDL, and MDA-LDL was measured by ELISA, and the ratio of antigen-specific IgM/total IgM was calculated. Shown are the mean ± SEM results of four samples. *** P

Techniques Used: Multiple Displacement Amplification, Isolation, Staining, Flow Cytometry, Cytometry, Binding Assay, Enzyme-linked Immunosorbent Assay

MDA-carrying MPs are increased at the culprit lesion site in STE-MI. A: Total numbers of MPs were quantified by flow cytometry in plasma isolated from peripheral (femoral artery) and coronary blood samples of patients with STE-MI. B–D: Cellular origin: Isolated MPs were stained with anti-CD41a (platelet), anti-CD235a (RBCs), and anti-CD31 (ECs) to identify their cellular origin and are presented as percentages of positive MPs of total MPs. E–H: MDA epitopes: Isolated MPs were stained with annexin V, anti-CD41a, or anti-CD235a and LR04, and analyzed by flow cytometry. Data show total numbers of annexin V + LR04 + MP per µl plasma (E) and percentages of annexin V + LR04 + MPs of total MPs (F), as well as the percentages of LR04 + MPs of either CD41a + platelet (G) or CD235a + RBC (H) MPs. All results are presented as mean ± SEM of 13–14 STE-MI patients; * P
Figure Legend Snippet: MDA-carrying MPs are increased at the culprit lesion site in STE-MI. A: Total numbers of MPs were quantified by flow cytometry in plasma isolated from peripheral (femoral artery) and coronary blood samples of patients with STE-MI. B–D: Cellular origin: Isolated MPs were stained with anti-CD41a (platelet), anti-CD235a (RBCs), and anti-CD31 (ECs) to identify their cellular origin and are presented as percentages of positive MPs of total MPs. E–H: MDA epitopes: Isolated MPs were stained with annexin V, anti-CD41a, or anti-CD235a and LR04, and analyzed by flow cytometry. Data show total numbers of annexin V + LR04 + MP per µl plasma (E) and percentages of annexin V + LR04 + MPs of total MPs (F), as well as the percentages of LR04 + MPs of either CD41a + platelet (G) or CD235a + RBC (H) MPs. All results are presented as mean ± SEM of 13–14 STE-MI patients; * P

Techniques Used: Multiple Displacement Amplification, Flow Cytometry, Cytometry, Isolation, Staining

19) Product Images from "Pulmonary Macrophage Transplantation Therapy"

Article Title: Pulmonary Macrophage Transplantation Therapy

Journal: Nature

doi: 10.1038/nature13807

Validation of Csf2rb-/- (KO) mice as an authentic model of human hPAP ( a ) Typical lung pathology showing surfactant-filled alveoli with well-preserved septa in a child homozygous for CSF2RB S271L mutations and identical pulmonary histopathology in a KO mouse. PAS stain. Scale bar, 100 μm. ( b ) Photographs of ‘milky’-appearing BAL from a 14 month-old KO mouse and normal-appearing BAL from an age-matched WT mouse (representative of n=6 mice/group). ( c ) Increased BAL turbidity and SP-D concentration in 4 month-old mice KO compared to age-matched WT mice. ( d ) BAL fluid biomarkers of hPAP (GM-CSF, M-CSF, and MCP-1) are reduced in 4 month-old KO mice compared to age-matched WT mice. ( e ) Alveolar macrophage biomarkers (PU.1, Pparg, Abcg1 mRNA) are reduced in 4 month-old KO compared to age-matched WT mice. ( f ) Progressive increase in BAL turbidity in KO mice but not age-matched WT mice (linear regression: KO, slope = 0.1271 ± 0.16 (r 2 , 0.311); WT, slope =0.031± 0.005). ( g ) Progressive increase in BAL fluid GM-CSF level in KO mice but not age-matched WT mice (linear regression: KO, slope =0.89±0.016 (r 2 , 0.249); WT, slope =0). Data are mean ± SEM of n=7 mice/group ( c-j ) or symbols representing individual WT (n=38) or KO (n=84) mice and the regression fit ± 95% CI lines. ( h ) GM-CSF bioactivity in BAL fluid from 10 month-old KO or WT mice (or 1 ng/ml murine GM- CSF) measured in the presence of anti-GM-CSF antibody (GM-CSF Ab) or isotype control (Control Ab) using the GM-CSF-stimulated STAT5 phosphorylation index (STAT5-PI) assay. Data are mean ± SEM of n=7 mice/group ( c-e ), n=4 ( h ) or symbols representing individual WT (n=38) or KO (n-84) mice and regression fit ± 95% CI ( f-g ). *p
Figure Legend Snippet: Validation of Csf2rb-/- (KO) mice as an authentic model of human hPAP ( a ) Typical lung pathology showing surfactant-filled alveoli with well-preserved septa in a child homozygous for CSF2RB S271L mutations and identical pulmonary histopathology in a KO mouse. PAS stain. Scale bar, 100 μm. ( b ) Photographs of ‘milky’-appearing BAL from a 14 month-old KO mouse and normal-appearing BAL from an age-matched WT mouse (representative of n=6 mice/group). ( c ) Increased BAL turbidity and SP-D concentration in 4 month-old mice KO compared to age-matched WT mice. ( d ) BAL fluid biomarkers of hPAP (GM-CSF, M-CSF, and MCP-1) are reduced in 4 month-old KO mice compared to age-matched WT mice. ( e ) Alveolar macrophage biomarkers (PU.1, Pparg, Abcg1 mRNA) are reduced in 4 month-old KO compared to age-matched WT mice. ( f ) Progressive increase in BAL turbidity in KO mice but not age-matched WT mice (linear regression: KO, slope = 0.1271 ± 0.16 (r 2 , 0.311); WT, slope =0.031± 0.005). ( g ) Progressive increase in BAL fluid GM-CSF level in KO mice but not age-matched WT mice (linear regression: KO, slope =0.89±0.016 (r 2 , 0.249); WT, slope =0). Data are mean ± SEM of n=7 mice/group ( c-j ) or symbols representing individual WT (n=38) or KO (n=84) mice and the regression fit ± 95% CI lines. ( h ) GM-CSF bioactivity in BAL fluid from 10 month-old KO or WT mice (or 1 ng/ml murine GM- CSF) measured in the presence of anti-GM-CSF antibody (GM-CSF Ab) or isotype control (Control Ab) using the GM-CSF-stimulated STAT5 phosphorylation index (STAT5-PI) assay. Data are mean ± SEM of n=7 mice/group ( c-e ), n=4 ( h ) or symbols representing individual WT (n=38) or KO (n-84) mice and regression fit ± 95% CI ( f-g ). *p

Techniques Used: Mouse Assay, Histopathology, Staining, Concentration Assay

Effects of PMT of gene-corrected macrophages on hPAP. ( a ) Macrophages derived from KO LSK cells transduced with GM-R-LV or GFP-LV, or from non-transduced WT LSK cells (indicated) were examined by light microscopy after Diff-Quick staining (top), or by immunofluorescence microscopy after staining with anti-CD131 (GM-CSF-R-β) and DAPI (upper middle), DAPI alone (lower middle), or anti-CD68 and DAPI (bottom). Images are representative of 3 experiments per condition. ( b ) Evaluation of GM-CSF receptor signaling in the indicated cells (before PMT) by measurement of GM-CSF-stimulated STAT5 phosphorylation by flow cytometry. Representative of n=3 experiments per condition. Quantitative summary data are shown in the manuscript ( Fig. 5b ). ( c ) Western blotting to detect GM-CSF receptor-β (CD131) (top) or actin (bottom, as a loading control) in BAL cells from age-matched KO mice 2 months after PMT as indicated (each lane represents one mouse of n=10, 8, 10/group, respectively). ( d ) Appearance of BAL from age-matched KO mice 2 months after PMT as indicated (representative of n=10, 8, 10/group, respectively). ( e-f ) One year after PMT of GM-R-LV transduced KO LSK cell-derived macrophages in KO mice, GFP + cells were identified ( e ) and evaluated for cell surface markers by flow cytometry ( f ) (representative of n=7 mice).
Figure Legend Snippet: Effects of PMT of gene-corrected macrophages on hPAP. ( a ) Macrophages derived from KO LSK cells transduced with GM-R-LV or GFP-LV, or from non-transduced WT LSK cells (indicated) were examined by light microscopy after Diff-Quick staining (top), or by immunofluorescence microscopy after staining with anti-CD131 (GM-CSF-R-β) and DAPI (upper middle), DAPI alone (lower middle), or anti-CD68 and DAPI (bottom). Images are representative of 3 experiments per condition. ( b ) Evaluation of GM-CSF receptor signaling in the indicated cells (before PMT) by measurement of GM-CSF-stimulated STAT5 phosphorylation by flow cytometry. Representative of n=3 experiments per condition. Quantitative summary data are shown in the manuscript ( Fig. 5b ). ( c ) Western blotting to detect GM-CSF receptor-β (CD131) (top) or actin (bottom, as a loading control) in BAL cells from age-matched KO mice 2 months after PMT as indicated (each lane represents one mouse of n=10, 8, 10/group, respectively). ( d ) Appearance of BAL from age-matched KO mice 2 months after PMT as indicated (representative of n=10, 8, 10/group, respectively). ( e-f ) One year after PMT of GM-R-LV transduced KO LSK cell-derived macrophages in KO mice, GFP + cells were identified ( e ) and evaluated for cell surface markers by flow cytometry ( f ) (representative of n=7 mice).

Techniques Used: Derivative Assay, Transduction, Light Microscopy, Diff-Quik, Staining, Immunofluorescence, Microscopy, Flow Cytometry, Cytometry, Western Blot, Mouse Assay

Pharmacokinetics and pharmacodynamics of PMT in KO mice. ( a ) Competitive proliferation of WT and KO BMDMs co-cultured with GM-CSF and M-CSF (n=3 plates/point). ( b ) Quantification of GFP + BAL cells 2 months after PMT of Lys-M GFP BMDMs into WT (n=3) or KO (n=6) mice. ( c ) Quantitation of Ki67 + Lys-M GFP cells in KO mice (n=3) one or twelve months after PMT. ( d-f ) KO mice received PMT of WT BMDMs and were evaluated at the indicated times to quantify CD131+BAL cells ( d ), BAL GM-CSF concentration ( e ), and BAL turbidity ( f ). Exponential regression (± prediction bands), R 2 =0.943 ( d ), R 2 =0.819 ( e ), R 2 =0.958 ( f ). Data are mean ± SEM for 3-7 mice/group. ( g ) Csf2rb mRNA in BAL cells from KO mice one year after PMT, or untreated, age-matched control mice (n=6). ( h ) Number of BAL cells (open bars) or CD131 + alveolar macrophages (closed bars) in KO mice one year after PMT (n = 5) or untreated WT mice (n=10). Data are mean ± SEM. *p
Figure Legend Snippet: Pharmacokinetics and pharmacodynamics of PMT in KO mice. ( a ) Competitive proliferation of WT and KO BMDMs co-cultured with GM-CSF and M-CSF (n=3 plates/point). ( b ) Quantification of GFP + BAL cells 2 months after PMT of Lys-M GFP BMDMs into WT (n=3) or KO (n=6) mice. ( c ) Quantitation of Ki67 + Lys-M GFP cells in KO mice (n=3) one or twelve months after PMT. ( d-f ) KO mice received PMT of WT BMDMs and were evaluated at the indicated times to quantify CD131+BAL cells ( d ), BAL GM-CSF concentration ( e ), and BAL turbidity ( f ). Exponential regression (± prediction bands), R 2 =0.943 ( d ), R 2 =0.819 ( e ), R 2 =0.958 ( f ). Data are mean ± SEM for 3-7 mice/group. ( g ) Csf2rb mRNA in BAL cells from KO mice one year after PMT, or untreated, age-matched control mice (n=6). ( h ) Number of BAL cells (open bars) or CD131 + alveolar macrophages (closed bars) in KO mice one year after PMT (n = 5) or untreated WT mice (n=10). Data are mean ± SEM. *p

Techniques Used: Mouse Assay, Cell Culture, Quantitation Assay, Concentration Assay

Effects of PMT of gene-corrected macrophages on hPAP severity and biomarkers. KO mice received PMT of non-transduced WT or LV-transduced KO macrophages and were evaluated after two month (2M) or one year (1Y) (with untreated, age-matched KO mice). Key indicates PMT cells used, prior LV treatment, and time after PMT analysis was performed. ( a ) LV schematics. ( b ) GM-CSF signaling measured by the STAT5 phosphorylation index (STAT5-PI) in the indicated cells before PMT. ( c ) BAL turbidity and SP-D concentration. ( d ) BAL biomarkers. Mean ± SEM of n=3 ( b ) or 5-10 ( c-g ) mice/group. *p
Figure Legend Snippet: Effects of PMT of gene-corrected macrophages on hPAP severity and biomarkers. KO mice received PMT of non-transduced WT or LV-transduced KO macrophages and were evaluated after two month (2M) or one year (1Y) (with untreated, age-matched KO mice). Key indicates PMT cells used, prior LV treatment, and time after PMT analysis was performed. ( a ) LV schematics. ( b ) GM-CSF signaling measured by the STAT5 phosphorylation index (STAT5-PI) in the indicated cells before PMT. ( c ) BAL turbidity and SP-D concentration. ( d ) BAL biomarkers. Mean ± SEM of n=3 ( b ) or 5-10 ( c-g ) mice/group. *p

Techniques Used: Mouse Assay, Concentration Assay

20) Product Images from "Inhibition of SREBP1 sensitizes cells to death ligands"

Article Title: Inhibition of SREBP1 sensitizes cells to death ligands

Journal: Oncotarget

doi:

Effects of SREBP1 or FASN inhibition on levels of core components of the death receptor pathway of caspase activation and ceramide A) FADD, Caspase 8, Caspase 3 and Flip were studied in HeLa cells treated with increasing concentrations of Orlistat or infected with SREBP1 or Control shRNA. Actin was used as loading control. B) Membrane ceramide expression was analized by Flow Cytometry after increasing concentrations of Orlistat for 24 hours or after FASN, SREBP1 or Control silencing shRNA in HeLa cells. *p
Figure Legend Snippet: Effects of SREBP1 or FASN inhibition on levels of core components of the death receptor pathway of caspase activation and ceramide A) FADD, Caspase 8, Caspase 3 and Flip were studied in HeLa cells treated with increasing concentrations of Orlistat or infected with SREBP1 or Control shRNA. Actin was used as loading control. B) Membrane ceramide expression was analized by Flow Cytometry after increasing concentrations of Orlistat for 24 hours or after FASN, SREBP1 or Control silencing shRNA in HeLa cells. *p

Techniques Used: Inhibition, Activation Assay, Infection, shRNA, Expressing, Flow Cytometry, Cytometry

21) Product Images from "MBL-Mediated Opsonophagocytosis of Candida albicans by Human Neutrophils Is Coupled with Intracellular Dectin-1-Triggered ROS Production"

Article Title: MBL-Mediated Opsonophagocytosis of Candida albicans by Human Neutrophils Is Coupled with Intracellular Dectin-1-Triggered ROS Production

Journal: PLoS ONE

doi: 10.1371/journal.pone.0050589

The inhibited phagocytic efficiency of human neutrophils by blockage of Dectin-1 was compensated by exogenous MBL. A. Abrogation effect of Dectin-1 on human neutrophils by 5 µg/mL blocking mAb was measured by flow cytometry. PE-mouse IgG2b was used as isotype control. B and C. Neutrophils containing intracellular FITC- C. albicans had distinctive green fluorescence, and were easily differentiated from the ones without intracellular FITC- C. albicans . Accordingly, the phagocytic efficiency of neutrophils was measured by flow cytometry assay after stimulation with FITC- C. albicans for 30 and 60 min in the presence of 5 µg/mL Dectin-1 blocking mAb and exogenous MBL at a series of concentrations of 2.5, 5 and 10 µg/mL. D. Bar graph depicted the phagocytic efficiency of human neutrophils at 30 or 60 min after stimulation by FITC- C. albicans in the presence of 5 µg/mL Dectin-1 blocking mAb and exogenous MBL at a series of concentrations of 2.5, 5 and 10 µg/mL. Data were represented as mean ± SE (n = 20). * Significant (
Figure Legend Snippet: The inhibited phagocytic efficiency of human neutrophils by blockage of Dectin-1 was compensated by exogenous MBL. A. Abrogation effect of Dectin-1 on human neutrophils by 5 µg/mL blocking mAb was measured by flow cytometry. PE-mouse IgG2b was used as isotype control. B and C. Neutrophils containing intracellular FITC- C. albicans had distinctive green fluorescence, and were easily differentiated from the ones without intracellular FITC- C. albicans . Accordingly, the phagocytic efficiency of neutrophils was measured by flow cytometry assay after stimulation with FITC- C. albicans for 30 and 60 min in the presence of 5 µg/mL Dectin-1 blocking mAb and exogenous MBL at a series of concentrations of 2.5, 5 and 10 µg/mL. D. Bar graph depicted the phagocytic efficiency of human neutrophils at 30 or 60 min after stimulation by FITC- C. albicans in the presence of 5 µg/mL Dectin-1 blocking mAb and exogenous MBL at a series of concentrations of 2.5, 5 and 10 µg/mL. Data were represented as mean ± SE (n = 20). * Significant (

Techniques Used: Blocking Assay, Flow Cytometry, Cytometry, Fluorescence

MBL - pre-incubated C. albicans stimulated intracellular expression of Dectin-1 in human neutrophils. The expression of neutrophil Dectin-1 was measured by PE-anti-human Dectin-1 mAb using flow cytometry at indicated time points after stimulation by live or HK- C. albicans at a MOI of 10 which was pre-treated with 10 µg/mL MBL. Mouse IgG2b was used as an isotype control. A. C. albicans -stimulated neutrophils were fixed with 1% paraformaldehyde, and the expression of Dectin-1 was measured by flow cytometry. B. Bar graph depicted the expression of Dectin-1 in neutrophils which were fixed with 1% paraformaldehyde at indicated time points after stimulation. C. After C. albicans -stimulated neutrophils were permeabilized and fixed with BD Cytofix/cytoperm solution for measuring intracellular cytokines, the expression of Dectin-1 was measured by flow cytometry. D. Bar graph depicted the expression of Dectin-1 in neutrophils which were permeabilized and fixed with BD Cytofix/cytoperm solution at indicated time points after stimulation. Data were represented as mean ± SE (n = 20). ** Highly significant (
Figure Legend Snippet: MBL - pre-incubated C. albicans stimulated intracellular expression of Dectin-1 in human neutrophils. The expression of neutrophil Dectin-1 was measured by PE-anti-human Dectin-1 mAb using flow cytometry at indicated time points after stimulation by live or HK- C. albicans at a MOI of 10 which was pre-treated with 10 µg/mL MBL. Mouse IgG2b was used as an isotype control. A. C. albicans -stimulated neutrophils were fixed with 1% paraformaldehyde, and the expression of Dectin-1 was measured by flow cytometry. B. Bar graph depicted the expression of Dectin-1 in neutrophils which were fixed with 1% paraformaldehyde at indicated time points after stimulation. C. After C. albicans -stimulated neutrophils were permeabilized and fixed with BD Cytofix/cytoperm solution for measuring intracellular cytokines, the expression of Dectin-1 was measured by flow cytometry. D. Bar graph depicted the expression of Dectin-1 in neutrophils which were permeabilized and fixed with BD Cytofix/cytoperm solution at indicated time points after stimulation. Data were represented as mean ± SE (n = 20). ** Highly significant (

Techniques Used: Incubation, Expressing, Flow Cytometry, Cytometry

Abrogation of Dectin-1 partly inhibited ROS production in neutrophils which was stimulated by C. albicans in the presence of MBL. A. Following the pretreatment with 0.1% Tween-20/PBS solution containing 10 µg/mL of MBL and 5 µg/mL of anti-human Dectin-1 blocking mAb or the mouse IgG2b (Isotype control), the maximum value of intracellular ROS in neutrophils stimulated by live C. albicans (MOI = 10) was determined by flow cytometry during 120 min. The neutrophils treated with PBS containing 10 µg/mL of MBL were set as the Tween-20 control. The expression of ROS was represented as mean fluorescence intensity (MFI). B. Bar graph depicted the maximum value of intracellular ROS during 120 min after stimulation by live or HK- C. albicans in the presence of 10 µg/mL of MBL and different dosages of Dectin-1 blocking mAb or the isotype. Data were represented as mean ± SE (n = 20). ** Highly significant (
Figure Legend Snippet: Abrogation of Dectin-1 partly inhibited ROS production in neutrophils which was stimulated by C. albicans in the presence of MBL. A. Following the pretreatment with 0.1% Tween-20/PBS solution containing 10 µg/mL of MBL and 5 µg/mL of anti-human Dectin-1 blocking mAb or the mouse IgG2b (Isotype control), the maximum value of intracellular ROS in neutrophils stimulated by live C. albicans (MOI = 10) was determined by flow cytometry during 120 min. The neutrophils treated with PBS containing 10 µg/mL of MBL were set as the Tween-20 control. The expression of ROS was represented as mean fluorescence intensity (MFI). B. Bar graph depicted the maximum value of intracellular ROS during 120 min after stimulation by live or HK- C. albicans in the presence of 10 µg/mL of MBL and different dosages of Dectin-1 blocking mAb or the isotype. Data were represented as mean ± SE (n = 20). ** Highly significant (

Techniques Used: Blocking Assay, Flow Cytometry, Cytometry, Expressing, Fluorescence

Intracellular expression of Dectin-1 was co-distributed with stimulated ROS on the C. albicans which was pre-incubated with MBL and phagocytized by human neutrophils. A and B . Localization of Dectin-1 expression and stimulated ROS were determined using confocal microscopy in human neutrophils which phagocytized live C. albicans . The distribution of intracellular Dectin-1 was indicated by red arrows and ROS by green arrows. The neutrophils which phagocytized C. albicans spores were indicated by yellow arrows. The localization association of intracellular Dectin-1 and stimulated ROS was indicated by blue arrows in the merged fluorescence image. C. rhDectin-1 binding before the treatment with β -1,3-glucanase. D. rhDectin-1 binding after the treatment with β -1,3-glucanase. C and D . Exposure of β -1,3-glucan on the spores of C. albicans was confirmed by indirect immunofluorescence assay using hrDectin-1 and PE-anti-human Dectin-1 mAb, and β -1,3-glucanase digestion test. The binding of Dectin-1 on the cell wall of C. albicans was indicted by blue arrows in the merged image. The data represented 3 similar experiments. Scale bars, A, B and C. 10 µm; D. 20 µm.
Figure Legend Snippet: Intracellular expression of Dectin-1 was co-distributed with stimulated ROS on the C. albicans which was pre-incubated with MBL and phagocytized by human neutrophils. A and B . Localization of Dectin-1 expression and stimulated ROS were determined using confocal microscopy in human neutrophils which phagocytized live C. albicans . The distribution of intracellular Dectin-1 was indicated by red arrows and ROS by green arrows. The neutrophils which phagocytized C. albicans spores were indicated by yellow arrows. The localization association of intracellular Dectin-1 and stimulated ROS was indicated by blue arrows in the merged fluorescence image. C. rhDectin-1 binding before the treatment with β -1,3-glucanase. D. rhDectin-1 binding after the treatment with β -1,3-glucanase. C and D . Exposure of β -1,3-glucan on the spores of C. albicans was confirmed by indirect immunofluorescence assay using hrDectin-1 and PE-anti-human Dectin-1 mAb, and β -1,3-glucanase digestion test. The binding of Dectin-1 on the cell wall of C. albicans was indicted by blue arrows in the merged image. The data represented 3 similar experiments. Scale bars, A, B and C. 10 µm; D. 20 µm.

Techniques Used: Expressing, Incubation, Confocal Microscopy, Fluorescence, Binding Assay, Immunofluorescence

MBL - pre-incubated C. albicans stimulated mRNA expression of Dectin-1 in human neutrophils. A. Products obtained by using human Dectin-1 cDNA primers were sequenced and compared to the reference sequence in NCBI. B. Bar graph depicted the fold expression of neutrophil Dectin-1 mRNA at indicated time points after stimulation by live or HK- C. albicans at a MOI of 10 which was pre-incubated with 10 µg/mL MBL for 30 min at 37°C. Data were represented as mean ± SE (n = 20). ** Highly significant (P
Figure Legend Snippet: MBL - pre-incubated C. albicans stimulated mRNA expression of Dectin-1 in human neutrophils. A. Products obtained by using human Dectin-1 cDNA primers were sequenced and compared to the reference sequence in NCBI. B. Bar graph depicted the fold expression of neutrophil Dectin-1 mRNA at indicated time points after stimulation by live or HK- C. albicans at a MOI of 10 which was pre-incubated with 10 µg/mL MBL for 30 min at 37°C. Data were represented as mean ± SE (n = 20). ** Highly significant (P

Techniques Used: Incubation, Expressing, Sequencing

22) Product Images from "Differentiation and migration properties of human foetal umbilical cord perivascular cells: potential for lung repair"

Article Title: Differentiation and migration properties of human foetal umbilical cord perivascular cells: potential for lung repair

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/j.1582-4934.2010.01047.x

Representative flow cytometry analysis of expanded foetal HUCPC. After three passages of culture the cells ( n = 24) were detached with trypsin, washed and stained with directly labelled monoclonal antibodies (pink histograms) or exposed to isotype-matched non-immune directly labelled immunoglobulins (blue histograms). Foetal HUCPC were positive for CD146, PDGF-Rβ, α-SMA and NG2, while were negative for CD45, CD34 and CD56 confirming the main features of perivascular cells. Moreover they expressed the typical mesenchymal markers such as CD90, CD73, CD105, CD44, HLA-ABC and they were negative for CD133, CD144 and HLA-DR.
Figure Legend Snippet: Representative flow cytometry analysis of expanded foetal HUCPC. After three passages of culture the cells ( n = 24) were detached with trypsin, washed and stained with directly labelled monoclonal antibodies (pink histograms) or exposed to isotype-matched non-immune directly labelled immunoglobulins (blue histograms). Foetal HUCPC were positive for CD146, PDGF-Rβ, α-SMA and NG2, while were negative for CD45, CD34 and CD56 confirming the main features of perivascular cells. Moreover they expressed the typical mesenchymal markers such as CD90, CD73, CD105, CD44, HLA-ABC and they were negative for CD133, CD144 and HLA-DR.

Techniques Used: Flow Cytometry, Cytometry, Staining

23) Product Images from "Adherence to human lung microvascular endothelial cells (HMVEC-L) of Plasmodium vivax isolates from Colombia"

Article Title: Adherence to human lung microvascular endothelial cells (HMVEC-L) of Plasmodium vivax isolates from Colombia

Journal: Malaria Journal

doi: 10.1186/1475-2875-12-347

Average adherence of Pv -iEs to the ICAM-1 endothelial receptor. 5*10 4 parasites/well were added to HMVEC-L-TNF, HMVEC-L-TNF- antiCD54 mAb, or to the basal control HMVEC-L. The scatter plot shows adherence data is an average of the number of Pv -iEs/200HMVEC-L; Asterisks indicate statistical significance with respect to adhesion values, as follows: *0.05 > p > 0.01; **0.01 > p > 0.001; ***p
Figure Legend Snippet: Average adherence of Pv -iEs to the ICAM-1 endothelial receptor. 5*10 4 parasites/well were added to HMVEC-L-TNF, HMVEC-L-TNF- antiCD54 mAb, or to the basal control HMVEC-L. The scatter plot shows adherence data is an average of the number of Pv -iEs/200HMVEC-L; Asterisks indicate statistical significance with respect to adhesion values, as follows: *0.05 > p > 0.01; **0.01 > p > 0.001; ***p

Techniques Used:

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

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

Journal: PLoS ONE

doi: 10.1371/journal.pone.0082742

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

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

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

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

25) Product Images from "Characterization of the first-in-class T-cell-engaging bispecific single-chain antibody for targeted immunotherapy of solid tumors expressing the oncofetal protein claudin 6"

Article Title: Characterization of the first-in-class T-cell-engaging bispecific single-chain antibody for targeted immunotherapy of solid tumors expressing the oncofetal protein claudin 6

Journal: Oncoimmunology

doi: 10.1080/2162402X.2015.1091555

6PHU3 mediates strong T-cell-engaging effects only in the presence of target cells. (A) Microscopic image after 24 h of co-culturing CLDN6 + PA-1/luc cells and CLDN6 – MDA-MB-231/luc control cells in an E:T ratio of 5:1 +/− 30 ng/mL 6PHU3 or ctrl bi-(scFv) 2 as indicated (200× magnification). Arrowheads: clusters of T cells on residual target cells (B) Activation of CD3 + T cells as analyzed by flow cytometry. T cells were incubated with the indicated 6PHU3 concentrations +/− PA-1 cells for 48 h. Percentages of total activated T cells (black columns), CD69 + (gray columns) and CD25 + (white columns) are shown. Ns signifies not significant ( p = 0.053); * p
Figure Legend Snippet: 6PHU3 mediates strong T-cell-engaging effects only in the presence of target cells. (A) Microscopic image after 24 h of co-culturing CLDN6 + PA-1/luc cells and CLDN6 – MDA-MB-231/luc control cells in an E:T ratio of 5:1 +/− 30 ng/mL 6PHU3 or ctrl bi-(scFv) 2 as indicated (200× magnification). Arrowheads: clusters of T cells on residual target cells (B) Activation of CD3 + T cells as analyzed by flow cytometry. T cells were incubated with the indicated 6PHU3 concentrations +/− PA-1 cells for 48 h. Percentages of total activated T cells (black columns), CD69 + (gray columns) and CD25 + (white columns) are shown. Ns signifies not significant ( p = 0.053); * p

Techniques Used: Multiple Displacement Amplification, Activation Assay, Flow Cytometry, Cytometry, Incubation

26) Product Images from "Genetic Diversity, Compartmentalization, and Age of HIV Proviruses Persisting in CD4+ T Cell Subsets during Long-Term Combination Antiretroviral Therapy"

Article Title: Genetic Diversity, Compartmentalization, and Age of HIV Proviruses Persisting in CD4+ T Cell Subsets during Long-Term Combination Antiretroviral Therapy

Journal: Journal of Virology

doi: 10.1128/JVI.01786-19

Gating strategy for sorting of CD4 + T cell subsets. Representative flow cytometry plot illustrating how participant CD4 + T cells were isolated after staining with the indicated antibodies. CD4 T cell subsets were defined as follows: T N , CD45RA + CCR7 + CD27 + ; T CM , CD45RA − CCR7 + CD27 + ; T TM , CD45RA − CCR7 − CD27 + ; T EM , CD45RA − CCR7 − CD27 − .
Figure Legend Snippet: Gating strategy for sorting of CD4 + T cell subsets. Representative flow cytometry plot illustrating how participant CD4 + T cells were isolated after staining with the indicated antibodies. CD4 T cell subsets were defined as follows: T N , CD45RA + CCR7 + CD27 + ; T CM , CD45RA − CCR7 + CD27 + ; T TM , CD45RA − CCR7 − CD27 + ; T EM , CD45RA − CCR7 − CD27 − .

Techniques Used: Flow Cytometry, Isolation, Staining

27) Product Images from "A tumor-intrinsic PD-L1/NLRP3 inflammasome signaling pathway drives resistance to anti–PD-1 immunotherapy"

Article Title: A tumor-intrinsic PD-L1/NLRP3 inflammasome signaling pathway drives resistance to anti–PD-1 immunotherapy

Journal: The Journal of Clinical Investigation

doi: 10.1172/JCI133055

PMN-MDSC accumulation contributes to tumor progression following anti–PD-1 Ab immunotherapy. ( A ) Schematic overview of the adaptive resistance pathway. ( B ) RNA-Seq differential gene expression analysis of tumor tissues following treatment of the autochthonous BRAF V600E PTEN –/– melanoma model with anti–PD-1 Ab therapy versus IgG isotype control (Ctrl) ( n = 3). ( C ) qRT-PCR analysis of target genes of interest in serial tumor fine-needle aspiration (FNA) biopsy specimens harvested from the transgenic BRAF V600E PTEN –/– melanoma model treated with anti–PD-1 Ab versus IgG isotype control ( n = 5). ( D ) Gr-1 immunohistochemical analysis of transgenic BRAF V600E PTEN –/– melanoma tissues following treatment with anti–PD-1 Ab versus IgG isotype control. Original magnification, ×40. Gr-1 staining is shown in red. Images are representative of 3 tumors per group. ( E ) PMN-MDSC flow cytometric analysis of transgenic BRAF V600E PTEN –/– melanoma tissues following treatment with anti–PD-1 Ab versus IgG isotype control. PMN-MDSCs were defined as live + CD45 + CD11b + Ly6G + Ly6C int F4/80 – cells. Shown are a representative flow dot plot and quantification graph of PMN-MDSC flow cytometric data ( n = 5). ( F ) qRT-PCR analysis of CXCR2 ligands in BRAF V600E PTEN –/– melanoma tissues treated with anti–PD-1 Ab following CD8 + T cell ablation in vivo ( n = 3). ( G ) In vivo tumor study of BRAF V600E PTEN –/– melanoma genetically silenced for CXCL5. Quantitation of tumor-infiltrating PMN-MDSCs by flow cytometry is shown along with an in vivo tumor growth curve of CXCL5-silenced BRAF V600E PTEN –/– melanoma versus BRAF V600E PTEN –/– NTC melanoma control tumors treated with anti–PD-1 Ab. Data were normalized to tumors treated with IgG isotype control ( n = 5). ( H ) Combination treatment with anti–PD-1 Ab and CXCR2 inhibitor (CXCR2i) in an in vivo BRAF V600E PTEN –/– melanoma study ( n = 5). Graphs show flow cytometric analysis of tumor-infiltrating PMN-MDSCs and live + CD45 + CD3 + CD8 + T cells. * P
Figure Legend Snippet: PMN-MDSC accumulation contributes to tumor progression following anti–PD-1 Ab immunotherapy. ( A ) Schematic overview of the adaptive resistance pathway. ( B ) RNA-Seq differential gene expression analysis of tumor tissues following treatment of the autochthonous BRAF V600E PTEN –/– melanoma model with anti–PD-1 Ab therapy versus IgG isotype control (Ctrl) ( n = 3). ( C ) qRT-PCR analysis of target genes of interest in serial tumor fine-needle aspiration (FNA) biopsy specimens harvested from the transgenic BRAF V600E PTEN –/– melanoma model treated with anti–PD-1 Ab versus IgG isotype control ( n = 5). ( D ) Gr-1 immunohistochemical analysis of transgenic BRAF V600E PTEN –/– melanoma tissues following treatment with anti–PD-1 Ab versus IgG isotype control. Original magnification, ×40. Gr-1 staining is shown in red. Images are representative of 3 tumors per group. ( E ) PMN-MDSC flow cytometric analysis of transgenic BRAF V600E PTEN –/– melanoma tissues following treatment with anti–PD-1 Ab versus IgG isotype control. PMN-MDSCs were defined as live + CD45 + CD11b + Ly6G + Ly6C int F4/80 – cells. Shown are a representative flow dot plot and quantification graph of PMN-MDSC flow cytometric data ( n = 5). ( F ) qRT-PCR analysis of CXCR2 ligands in BRAF V600E PTEN –/– melanoma tissues treated with anti–PD-1 Ab following CD8 + T cell ablation in vivo ( n = 3). ( G ) In vivo tumor study of BRAF V600E PTEN –/– melanoma genetically silenced for CXCL5. Quantitation of tumor-infiltrating PMN-MDSCs by flow cytometry is shown along with an in vivo tumor growth curve of CXCL5-silenced BRAF V600E PTEN –/– melanoma versus BRAF V600E PTEN –/– NTC melanoma control tumors treated with anti–PD-1 Ab. Data were normalized to tumors treated with IgG isotype control ( n = 5). ( H ) Combination treatment with anti–PD-1 Ab and CXCR2 inhibitor (CXCR2i) in an in vivo BRAF V600E PTEN –/– melanoma study ( n = 5). Graphs show flow cytometric analysis of tumor-infiltrating PMN-MDSCs and live + CD45 + CD3 + CD8 + T cells. * P

Techniques Used: RNA Sequencing Assay, Expressing, Quantitative RT-PCR, Transgenic Assay, Immunohistochemistry, Staining, In Vivo, Quantitation Assay, Flow Cytometry

28) Product Images from "Evaluation of epithelial progenitor cells and growth factors in a preclinical model of wound healing induced by mesenchymal stromal cells"

Article Title: Evaluation of epithelial progenitor cells and growth factors in a preclinical model of wound healing induced by mesenchymal stromal cells

Journal: Bioscience Reports

doi: 10.1042/BSR20200461

Phenotypical and functional characterization of MSC Microscopical observation shows the fibroblast-like morphology of MSC in culture ( A ). Flow cytometry analysis of MSC marker expression shows the expression of CD73 and CD90 (arrows). Negative controls were stained with the respective isotype (arrows) ( B ). Multipotent differentiation assays show the osteogenic ( C ), adipogenic ( D ) and chondrogenic ( E ) potential of MSC.
Figure Legend Snippet: Phenotypical and functional characterization of MSC Microscopical observation shows the fibroblast-like morphology of MSC in culture ( A ). Flow cytometry analysis of MSC marker expression shows the expression of CD73 and CD90 (arrows). Negative controls were stained with the respective isotype (arrows) ( B ). Multipotent differentiation assays show the osteogenic ( C ), adipogenic ( D ) and chondrogenic ( E ) potential of MSC.

Techniques Used: Functional Assay, Flow Cytometry, Marker, Expressing, Staining

29) Product Images from "Automated application of low energy electron irradiation enables inactivation of pathogen- and cell-containing liquids in biomedical research and production facilities"

Article Title: Automated application of low energy electron irradiation enables inactivation of pathogen- and cell-containing liquids in biomedical research and production facilities

Journal: Scientific Reports

doi: 10.1038/s41598-020-69347-7

Electron beam irradiation causes a dose-dependent decrease of cell number, cell viability and cytotoxicity of NK-92 cells. ( A ) NK-92 cells were irradiated at 2.2 Gy (blue, n = 3), 6.6 Gy (red, n = 2) or 11 Gy grey, n = 4) and compared to non-irradiated control cells (black, n = 4) for growth, viability and activity against K562 cells (Effector:Target-ratio 5:1) during 3 days after irradiation. Values are displayed as mean ± standard deviation. ( B ) Surface expression of CD56 in NK-92 cells was analyzed by flow cytometry 2 days post irradiation. Mean fluorescence intensity (MFI) was compared to non-irradiated NK-92 cells. Mean values (n = 3) ± SD are shown, the non-irradiated control was set to 100%.
Figure Legend Snippet: Electron beam irradiation causes a dose-dependent decrease of cell number, cell viability and cytotoxicity of NK-92 cells. ( A ) NK-92 cells were irradiated at 2.2 Gy (blue, n = 3), 6.6 Gy (red, n = 2) or 11 Gy grey, n = 4) and compared to non-irradiated control cells (black, n = 4) for growth, viability and activity against K562 cells (Effector:Target-ratio 5:1) during 3 days after irradiation. Values are displayed as mean ± standard deviation. ( B ) Surface expression of CD56 in NK-92 cells was analyzed by flow cytometry 2 days post irradiation. Mean fluorescence intensity (MFI) was compared to non-irradiated NK-92 cells. Mean values (n = 3) ± SD are shown, the non-irradiated control was set to 100%.

Techniques Used: Irradiation, Activity Assay, Standard Deviation, Expressing, Flow Cytometry, Fluorescence

30) Product Images from "CCR5 deficiency impairs CD4+ T‐cell memory responses and antigenic sensitivity through increased ceramide synthesis"

Article Title: CCR5 deficiency impairs CD4+ T‐cell memory responses and antigenic sensitivity through increased ceramide synthesis

Journal: The EMBO Journal

doi: 10.15252/embj.2020104749

CCR 5 deficiency impairs CD 4 + T‐cell memory responses Representative plots of splenocytes from CD45.1 mice adoptively transferred with CD45.2 OT‐II WT or CCR5 −/− lymph node cell suspensions, 5 weeks after infection with rVACV‐OVA virus. The gating strategy used to identify the memory CD4 + T‐cell subtypes is shown ( n = 5). Absolute number of OT‐II cells recovered in spleens of mice as in A ( n = 5). Percentage of CD4 + T EM (C) and T CM (D) in the OT‐II WT and CCR5 −/− populations ( n = 5). IFNγ‐producing OT‐II WT and CCR5 −/− memory cells isolated from mice as in (A) and restimulated ex vivo with OVA 323–339 (1 μM) ( n = 4). Immunization scheme for NIP‐OVA and NIP‐KLH in WT and CCR5 −/− mice. Representative plots (G) and quantification of the frequency (H) and absolute number (I) of T fh cells (CD4 + CD44 + PD‐1 + CXCR5 + ) in the spleen after primary immunization (day 7) with NIP‐OVA ( n = 7). ELISA analysis of high‐ (J) and low‐affinity (K) isotype‐specific anti‐NIP antibodies in sera from OVA/OVA‐ and OVA/KLH‐immunized mice (day 15 post‐challenge; n = 5 mice/group). Data representative of one experiment of two. Data information: (B–E, H–K), Data are mean ± SEM. * P
Figure Legend Snippet: CCR 5 deficiency impairs CD 4 + T‐cell memory responses Representative plots of splenocytes from CD45.1 mice adoptively transferred with CD45.2 OT‐II WT or CCR5 −/− lymph node cell suspensions, 5 weeks after infection with rVACV‐OVA virus. The gating strategy used to identify the memory CD4 + T‐cell subtypes is shown ( n = 5). Absolute number of OT‐II cells recovered in spleens of mice as in A ( n = 5). Percentage of CD4 + T EM (C) and T CM (D) in the OT‐II WT and CCR5 −/− populations ( n = 5). IFNγ‐producing OT‐II WT and CCR5 −/− memory cells isolated from mice as in (A) and restimulated ex vivo with OVA 323–339 (1 μM) ( n = 4). Immunization scheme for NIP‐OVA and NIP‐KLH in WT and CCR5 −/− mice. Representative plots (G) and quantification of the frequency (H) and absolute number (I) of T fh cells (CD4 + CD44 + PD‐1 + CXCR5 + ) in the spleen after primary immunization (day 7) with NIP‐OVA ( n = 7). ELISA analysis of high‐ (J) and low‐affinity (K) isotype‐specific anti‐NIP antibodies in sera from OVA/OVA‐ and OVA/KLH‐immunized mice (day 15 post‐challenge; n = 5 mice/group). Data representative of one experiment of two. Data information: (B–E, H–K), Data are mean ± SEM. * P

Techniques Used: Mouse Assay, Infection, Isolation, Ex Vivo, Enzyme-linked Immunosorbent Assay

31) Product Images from "HLF Expression Defines the Human Haematopoietic Stem Cell State"

Article Title: HLF Expression Defines the Human Haematopoietic Stem Cell State

Journal: bioRxiv

doi: 10.1101/2020.06.29.177709

Specific Expression of HLF expression in enriched human HSC populations. a) HLF expression is enriched in cultured human HSC subsets. Differentially expressed genes from CD201+ 7 and ITGA3/CD201+ 21 HSC-enriched population transcriptomes were intersected to identify consistently up or downregulated genes. Ranking based on fold-change between the most enriched (ITGA3 + /CD201 + ) and most depleted (CD201-) HSC populations is summarized by a waterfall plot (left, log2-transformed). Range of expression is provided for each gene in square brackets (TPM = transcripts per kilobase million). b) HLF-expression is undetectable in blood leukocyte populations. (curated from 22 ). c) cell transcriptomic overview of cord blood cell populations. Fresh and UM171-expanded CD34 + (d7) (2 samples each condition, 15921 cells total), were scRNA sequenced (10X Chromium), integrated and clustered using Seurat 3 43 ). Ten cell clusters were identified: haematopoietic stem/progenitor cells (HSPC), lymphoid-primed multipotent progenitors (LMPP), multipotent progenitors (MPP), granulo-monocytic progenitor (GMP), megakaryocyte-erythroid-mast cell progenitors (MEMP), megakaryocytes (mega), eosinophil/basophils (eo/ba), mast cells (mast), erythroid lineage cells, neutrophils (neutro) and monocytic/dendritic cells (mono/dc). Dimensional reduction was calculated using SPRING 44 . d-e) HSPC specific HLF expression in fresh CD34 + cells and after 7d ex vivo expansion in the presence of UM171. HLF expression is shown in single cell transcriptomes split up by treatment. Normalized expression data (z-score, after MAGIC imputation 45 ) is expressed in color scale. f) Comparison of HLF expression specificity versus selected HSC-associated genes and common HSC surface marker genes. Gene-wise z-score distribution by treatment (fresh CD34+ and UM171 d7) is represented as density for each cell community (same color-code as in Fig. 1c ). Mean z-score for HLF in HSPC cluster is provided as dotted reference line for each treatment. g-i) HLF expression is strongly enriched in HSC clusters in human bone marrow. g) Overview of cell clusters. (Human Cell Atlas; 101,935 cells; integrated data from eight donors, UMAP reduction; preprocessed data, clusters and labels adopted from 32 ). h) HLF expression . (z-score normalized, after MAGIC imputation). i) Expression summary of selected HSC-associated genes in human bone marrow . (scaled expression averaged for each haematopoietic cell population and donor (n=8) in the dataset, row-normalized color scale). j-l) HLF expression is restricted to HSC/MPP cluster in haematopoietic human fetal liver cells. j) Overview of cell communities . (Human Cell Atlas; 113,063 cells; integrated data from 14 fetal livers across four developmental stages, UMAP reduction; preprocessed data, clusters and labels adopted from 33 ). Mac, macrophage; Neut-my, neutrophil–myeloid; Mono-mac, monocyte-macrophage; Early L/TL, early lymphoid/T lymphocyte; pro., progenitor. k) HLF expression (z-score normalized, after MAGIC imputation). I) Expression summary of HSC-associated genes in fetal liver (scaled expression was averaged for each haematopoietic cell population and four gestation stages (7-8, 9-11, 12-14 and 15-17 post-conception weeks), row-normalized color scale).
Figure Legend Snippet: Specific Expression of HLF expression in enriched human HSC populations. a) HLF expression is enriched in cultured human HSC subsets. Differentially expressed genes from CD201+ 7 and ITGA3/CD201+ 21 HSC-enriched population transcriptomes were intersected to identify consistently up or downregulated genes. Ranking based on fold-change between the most enriched (ITGA3 + /CD201 + ) and most depleted (CD201-) HSC populations is summarized by a waterfall plot (left, log2-transformed). Range of expression is provided for each gene in square brackets (TPM = transcripts per kilobase million). b) HLF-expression is undetectable in blood leukocyte populations. (curated from 22 ). c) cell transcriptomic overview of cord blood cell populations. Fresh and UM171-expanded CD34 + (d7) (2 samples each condition, 15921 cells total), were scRNA sequenced (10X Chromium), integrated and clustered using Seurat 3 43 ). Ten cell clusters were identified: haematopoietic stem/progenitor cells (HSPC), lymphoid-primed multipotent progenitors (LMPP), multipotent progenitors (MPP), granulo-monocytic progenitor (GMP), megakaryocyte-erythroid-mast cell progenitors (MEMP), megakaryocytes (mega), eosinophil/basophils (eo/ba), mast cells (mast), erythroid lineage cells, neutrophils (neutro) and monocytic/dendritic cells (mono/dc). Dimensional reduction was calculated using SPRING 44 . d-e) HSPC specific HLF expression in fresh CD34 + cells and after 7d ex vivo expansion in the presence of UM171. HLF expression is shown in single cell transcriptomes split up by treatment. Normalized expression data (z-score, after MAGIC imputation 45 ) is expressed in color scale. f) Comparison of HLF expression specificity versus selected HSC-associated genes and common HSC surface marker genes. Gene-wise z-score distribution by treatment (fresh CD34+ and UM171 d7) is represented as density for each cell community (same color-code as in Fig. 1c ). Mean z-score for HLF in HSPC cluster is provided as dotted reference line for each treatment. g-i) HLF expression is strongly enriched in HSC clusters in human bone marrow. g) Overview of cell clusters. (Human Cell Atlas; 101,935 cells; integrated data from eight donors, UMAP reduction; preprocessed data, clusters and labels adopted from 32 ). h) HLF expression . (z-score normalized, after MAGIC imputation). i) Expression summary of selected HSC-associated genes in human bone marrow . (scaled expression averaged for each haematopoietic cell population and donor (n=8) in the dataset, row-normalized color scale). j-l) HLF expression is restricted to HSC/MPP cluster in haematopoietic human fetal liver cells. j) Overview of cell communities . (Human Cell Atlas; 113,063 cells; integrated data from 14 fetal livers across four developmental stages, UMAP reduction; preprocessed data, clusters and labels adopted from 33 ). Mac, macrophage; Neut-my, neutrophil–myeloid; Mono-mac, monocyte-macrophage; Early L/TL, early lymphoid/T lymphocyte; pro., progenitor. k) HLF expression (z-score normalized, after MAGIC imputation). I) Expression summary of HSC-associated genes in fetal liver (scaled expression was averaged for each haematopoietic cell population and four gestation stages (7-8, 9-11, 12-14 and 15-17 post-conception weeks), row-normalized color scale).

Techniques Used: Expressing, Cell Culture, Transformation Assay, Ex Vivo, Marker

HLF-reporter labels repopulating cells in CD34+ cord blood cell cultures. a) FACS plots showing the sorting of HLF-reporter targeted population for transplantation. rAAV6 HLF-ZE : recombinant rAAV6 particle encoding an HLF repair template with ires ZsGreen P2A tEGFR cassette. b) Summary of transplantation layout. Transplantation cohorts and cell doses are represented using the same color-code as in (a). c) Human engraftment summary of transplanted NSGS recipients. Human bone marrow chimerism determined based on human CD45+ cells among total CD45+ (mouse and human) cells at short (week 3), intermediate (week 9) and long-term (week 16) post-transplantation timepoints is plotted using the same color code as in (a) and (b). Each recipient mouse is represented along the x-axis (NSGS-ID). Recipients are arranged by descending average reconstitution across all timepoints. Recipients #25912, #25914 and #25916 were sacrificed at week 10 post-transplantation to be used as donors for secondary transplantation (summarized in Fig. 5 ). d) Lineage proportion of transplanted recipients. Bone marrow biopsies were analyzed and are arranged along timepoints and the individual recipients as in (e). Normalized proportions of B-cells (CD19), myeloid cells (CD33) and T-cells (CD3) within human CD45 + cells for each timepoint and recipient are color-coded as indicated. e) HR allele frequencies in pre-transplanted cell populations. top panel: ddPCR droplets are pre-gated based on FAM-positivity, black droplets represent FAM+/HEX-events indicative of untargeted alleles, red droplets (FAM/HEX double positive) indicate targeted alleles, sub-sampled to 300 droplets per specimen. bottom panel, quantification summary of HR frequencies calculated based on targeted/(untargeted+targeted) droplets. f) ddPCR analysis of bone marrow biopsies at weeks 3, 9 and 16. Specimens are arranged as in (c), ddPCR droplets are represented as in (e), sub-sampled to 50 droplets per specimen and timepoint. g) HR allele tracing summary. Summarized data representation of (e) and (f). Dashed red lines represent allele frequencies at time of transplantation. Bars represent average HR allele frequencies from (e) with standard error bars, color-codes as in (a-c).
Figure Legend Snippet: HLF-reporter labels repopulating cells in CD34+ cord blood cell cultures. a) FACS plots showing the sorting of HLF-reporter targeted population for transplantation. rAAV6 HLF-ZE : recombinant rAAV6 particle encoding an HLF repair template with ires ZsGreen P2A tEGFR cassette. b) Summary of transplantation layout. Transplantation cohorts and cell doses are represented using the same color-code as in (a). c) Human engraftment summary of transplanted NSGS recipients. Human bone marrow chimerism determined based on human CD45+ cells among total CD45+ (mouse and human) cells at short (week 3), intermediate (week 9) and long-term (week 16) post-transplantation timepoints is plotted using the same color code as in (a) and (b). Each recipient mouse is represented along the x-axis (NSGS-ID). Recipients are arranged by descending average reconstitution across all timepoints. Recipients #25912, #25914 and #25916 were sacrificed at week 10 post-transplantation to be used as donors for secondary transplantation (summarized in Fig. 5 ). d) Lineage proportion of transplanted recipients. Bone marrow biopsies were analyzed and are arranged along timepoints and the individual recipients as in (e). Normalized proportions of B-cells (CD19), myeloid cells (CD33) and T-cells (CD3) within human CD45 + cells for each timepoint and recipient are color-coded as indicated. e) HR allele frequencies in pre-transplanted cell populations. top panel: ddPCR droplets are pre-gated based on FAM-positivity, black droplets represent FAM+/HEX-events indicative of untargeted alleles, red droplets (FAM/HEX double positive) indicate targeted alleles, sub-sampled to 300 droplets per specimen. bottom panel, quantification summary of HR frequencies calculated based on targeted/(untargeted+targeted) droplets. f) ddPCR analysis of bone marrow biopsies at weeks 3, 9 and 16. Specimens are arranged as in (c), ddPCR droplets are represented as in (e), sub-sampled to 50 droplets per specimen and timepoint. g) HR allele tracing summary. Summarized data representation of (e) and (f). Dashed red lines represent allele frequencies at time of transplantation. Bars represent average HR allele frequencies from (e) with standard error bars, color-codes as in (a-c).

Techniques Used: FACS, Transplantation Assay, Recombinant

Engineering of a human genomic HLF -reporter. a) Outline of the HLF-reporter targeting strategy using CRISPR/Cas9 and rAAV6. A site-specific DSB at the HLF stop codon (orange) located in exon 4 is generated by a Cas9/sgHLF ribonucleoprotein (RNP) complex. This stimulates homologous recombination (HR) with a single-stranded donor template delivered through rAAV6 infection. The resulting HR event results in a transgenic locus that co-expresses the HLF open reading frame and a multifunctional ZsGreen (ZsG) expression cassette connected the endogenous HLF open reading frame by an EMCV internal ribosome entry site (ires). Grey boxes, HLF exons; white boxes, 5’ and 3’ untranslated regions; 3’/5’ HA, homology arms; purple box, puromycin resistance or truncated EGFR (tEGFR) sequence linked to ZsG by a P2A for optional drug or antibody mediated selection; WPRE, Woodchuck Hepatitis Virus Post-transcriptional Response Element to improve transcript stability; pA, endogenous HLF polyadenylation signal. b-e) Validation of the HLF reporter in human cell lines. HepG2 (HLF-expressing) and HEK293 (HLF non-expressing) cells were electroporated with Cas9/sgRNA RNP either as summarized in (a) or using sgAAVS1 as control. HLF-ZP , rAAV6 encoded HLF repair template driving expression of ZsG and Puromycin resistance. b) Droplet digital PCR genotyping of targeted cell lines. Black dots represent HR-negative and red dots represent HR-positive PCR droplets. HR percentages (printed in red) were calculated as HR-positive divided by the total number of specific amplicon-containing droplets (black and red). c) ddPCR strategy. ext. FW, external forward primer binding to a common region outside the 5’ HA; 3’ RV (reverse) primer amplifying unrecombined locus; ires RV (reverse) primer amplifying recombined locus; HR-negative and positive amplicons are detected by a common FAM-labeled probe and HR-positive amplicons are additionally recognized by a HEX-labeled probe that binds to the ires region of the transgene. d) FACS analysis to detect reporter expression. e) HLF expression levels in selected cell lines. Data curated from Human Protein Atlas 46 . f) Outline of experimental strategy to optimize reporter functionality in CD34 + cord blood cells. g) FACS sorting strategy to enrich/deplete HSCs based on CD201 expression from expanded CD34 + cells at d3 of culture. h) Selective HLF-reporter expression in HSC-containing subfractions of CD34+ cord blood cell cultures. HR allele frequencies were determined from one of four replicate wells. Total and ZsG + cell counts were determined by FACS on d7 and are normalized to 10e4 cells plated per 96-well post-electroporation at d4. MOI, multiplicity-of-infection. i) Effect of TP53 knock-down on HR and cell survival. CD201 + cells were sorted and targeted as in (h) (MOI400), and as an additional condition, electroporated with RNP and siRNA against TP53.
Figure Legend Snippet: Engineering of a human genomic HLF -reporter. a) Outline of the HLF-reporter targeting strategy using CRISPR/Cas9 and rAAV6. A site-specific DSB at the HLF stop codon (orange) located in exon 4 is generated by a Cas9/sgHLF ribonucleoprotein (RNP) complex. This stimulates homologous recombination (HR) with a single-stranded donor template delivered through rAAV6 infection. The resulting HR event results in a transgenic locus that co-expresses the HLF open reading frame and a multifunctional ZsGreen (ZsG) expression cassette connected the endogenous HLF open reading frame by an EMCV internal ribosome entry site (ires). Grey boxes, HLF exons; white boxes, 5’ and 3’ untranslated regions; 3’/5’ HA, homology arms; purple box, puromycin resistance or truncated EGFR (tEGFR) sequence linked to ZsG by a P2A for optional drug or antibody mediated selection; WPRE, Woodchuck Hepatitis Virus Post-transcriptional Response Element to improve transcript stability; pA, endogenous HLF polyadenylation signal. b-e) Validation of the HLF reporter in human cell lines. HepG2 (HLF-expressing) and HEK293 (HLF non-expressing) cells were electroporated with Cas9/sgRNA RNP either as summarized in (a) or using sgAAVS1 as control. HLF-ZP , rAAV6 encoded HLF repair template driving expression of ZsG and Puromycin resistance. b) Droplet digital PCR genotyping of targeted cell lines. Black dots represent HR-negative and red dots represent HR-positive PCR droplets. HR percentages (printed in red) were calculated as HR-positive divided by the total number of specific amplicon-containing droplets (black and red). c) ddPCR strategy. ext. FW, external forward primer binding to a common region outside the 5’ HA; 3’ RV (reverse) primer amplifying unrecombined locus; ires RV (reverse) primer amplifying recombined locus; HR-negative and positive amplicons are detected by a common FAM-labeled probe and HR-positive amplicons are additionally recognized by a HEX-labeled probe that binds to the ires region of the transgene. d) FACS analysis to detect reporter expression. e) HLF expression levels in selected cell lines. Data curated from Human Protein Atlas 46 . f) Outline of experimental strategy to optimize reporter functionality in CD34 + cord blood cells. g) FACS sorting strategy to enrich/deplete HSCs based on CD201 expression from expanded CD34 + cells at d3 of culture. h) Selective HLF-reporter expression in HSC-containing subfractions of CD34+ cord blood cell cultures. HR allele frequencies were determined from one of four replicate wells. Total and ZsG + cell counts were determined by FACS on d7 and are normalized to 10e4 cells plated per 96-well post-electroporation at d4. MOI, multiplicity-of-infection. i) Effect of TP53 knock-down on HR and cell survival. CD201 + cells were sorted and targeted as in (h) (MOI400), and as an additional condition, electroporated with RNP and siRNA against TP53.

Techniques Used: CRISPR, Generated, Homologous Recombination, Infection, Transgenic Assay, Expressing, Sequencing, Selection, Digital PCR, Polymerase Chain Reaction, Amplification, Binding Assay, Labeling, FACS, Electroporation

HLF-reporter labels human HSCs with extensive self-renewal capacity. a) FACS plots of CD34+/HLF-ZsG+ population . Representative bone marrow biopsies of reporter-negative ( sgHLF/ rAAV6 HLF-ZE targeted, ZsG − sorted, left) and reporter-positive ( sgHLF/ rAAV6 HLF-ZE targeted, ZsG + sorted, right panel) primary recipients, gated on human CD45+. b) Summary of CD34+/HLF-ZsG+ population. Population overview of all primary recipients, pre-gated on human CD45+, recipient mice are arranged according to engraftment levels as in Fig. 4c . c) Strategy for secondary transplantation. Bone marrow of three primary recipients ( sgHLF/ rAAV6 HLF-ZE targeted, ZsG + sorted cohort) was pooled and magnetically enriched for human CD34 expression. Reporter-expressing (ZsG+) and non-expressing cells (ZsG-) with comparable levels of CD34 expression were sorted for transplantation. Intra-hepatic transplantation into newborn NSGS recipients as outlined. Corresponding cell doses of HLF-ZsG+ (n=10) and HLF-ZsG- (n=7) were transplanted. d) Human engraftment summary of secondary recipients. Human bone chimerism in indicated tissues was determined based on human CD45-expressing cells among total (mouse and human) CD45+ cells at short (week 5, blood), intermediate (week 9, marrow) and long-term (week 16, marrow and spleen) post-transplantation. Dashed line represents the 0.1% mark used as cut-off for engraftment positivity. Significance was calculated by unpaired, one-sided (alternative = “greater”) Wilcoxon test and is provided as p-value for a given comparison. d) Lineage distribution of engrafted human cells. Positive specimens from (d) are shown and color-coded for B-cells (CD19+), myeloid cells (CD33+) and T-cells (CD3). Normalized for lineage proportions within human CD45+ cells. Samples with less than 0.1% of human chimerism are designated negative (neg.).
Figure Legend Snippet: HLF-reporter labels human HSCs with extensive self-renewal capacity. a) FACS plots of CD34+/HLF-ZsG+ population . Representative bone marrow biopsies of reporter-negative ( sgHLF/ rAAV6 HLF-ZE targeted, ZsG − sorted, left) and reporter-positive ( sgHLF/ rAAV6 HLF-ZE targeted, ZsG + sorted, right panel) primary recipients, gated on human CD45+. b) Summary of CD34+/HLF-ZsG+ population. Population overview of all primary recipients, pre-gated on human CD45+, recipient mice are arranged according to engraftment levels as in Fig. 4c . c) Strategy for secondary transplantation. Bone marrow of three primary recipients ( sgHLF/ rAAV6 HLF-ZE targeted, ZsG + sorted cohort) was pooled and magnetically enriched for human CD34 expression. Reporter-expressing (ZsG+) and non-expressing cells (ZsG-) with comparable levels of CD34 expression were sorted for transplantation. Intra-hepatic transplantation into newborn NSGS recipients as outlined. Corresponding cell doses of HLF-ZsG+ (n=10) and HLF-ZsG- (n=7) were transplanted. d) Human engraftment summary of secondary recipients. Human bone chimerism in indicated tissues was determined based on human CD45-expressing cells among total (mouse and human) CD45+ cells at short (week 5, blood), intermediate (week 9, marrow) and long-term (week 16, marrow and spleen) post-transplantation. Dashed line represents the 0.1% mark used as cut-off for engraftment positivity. Significance was calculated by unpaired, one-sided (alternative = “greater”) Wilcoxon test and is provided as p-value for a given comparison. d) Lineage distribution of engrafted human cells. Positive specimens from (d) are shown and color-coded for B-cells (CD19+), myeloid cells (CD33+) and T-cells (CD3). Normalized for lineage proportions within human CD45+ cells. Samples with less than 0.1% of human chimerism are designated negative (neg.).

Techniques Used: FACS, Mouse Assay, Transplantation Assay, Expressing

Selective HLF-reporter expression in human cord blood derived LT-HSC populations. Cord blood derived CD34+ cells were processed as in Fig. 2f with the addition of siTP53 and transduction of rAAV6 HLF-ZsG P2A tEGFR at MOI400. a) HR allele frequencies in CD201 + / − pre-sorted fractions as determined by ddPCR. Gated on FAM+ (common probe) droplets, HEX+ droplets (red) identify HR allele amplicons. b-c) Reporter expression in CD201 + / − pre-sorted fractions. Aggregated FACS analysis (b) and summary by repeat (n=4 for CD201- and n=5 for CD201+ sorted, unpaired two-sided Wilcoxon test p-value is indicated) in (c). d-e) Immuno-phenotypes of ex vivo expanded (+UM171) HLF-targeted HSPCs. FACS analysis of total (black) versus reporter expressing (green) populations at day 7. Percentages of increasingly restricted HSC gates are provided for each population. Aggregated FACS data in (d) and summary by repeat in (e). f) Dimensional reduction based on FACS analysis. UMAP reduction using CD34, CD45RA, CD201, CD90, ITGA3 and ZsG FACS intensities from (d) was calculated and is represented as 2d density plot of all cells (grey, n = 306,797). Cells falling into HSC- or ZsG-gates are overlayed and color-coded as in (d). g-h) HLF-reporter expression in immuno-phenotypic HSC gates. Reverse gating of the same data as above showing reporter expression in increasingly restricted HSC gates. Aggregated FACS data from all repeats in (g), summary in (h). i) Extrapolated HLF expression within immuno-phenotypic HSC gates. Ranges of cell fractions expressing HLF are summarized for each sub-population under consideration of measured allelic HR ratios of 20.2% (a). Lower boundaries are based on a scenario where all ZsG+ cells carry bi-allelic reporter integration, upper values are based on entirely mono-allelic integration.
Figure Legend Snippet: Selective HLF-reporter expression in human cord blood derived LT-HSC populations. Cord blood derived CD34+ cells were processed as in Fig. 2f with the addition of siTP53 and transduction of rAAV6 HLF-ZsG P2A tEGFR at MOI400. a) HR allele frequencies in CD201 + / − pre-sorted fractions as determined by ddPCR. Gated on FAM+ (common probe) droplets, HEX+ droplets (red) identify HR allele amplicons. b-c) Reporter expression in CD201 + / − pre-sorted fractions. Aggregated FACS analysis (b) and summary by repeat (n=4 for CD201- and n=5 for CD201+ sorted, unpaired two-sided Wilcoxon test p-value is indicated) in (c). d-e) Immuno-phenotypes of ex vivo expanded (+UM171) HLF-targeted HSPCs. FACS analysis of total (black) versus reporter expressing (green) populations at day 7. Percentages of increasingly restricted HSC gates are provided for each population. Aggregated FACS data in (d) and summary by repeat in (e). f) Dimensional reduction based on FACS analysis. UMAP reduction using CD34, CD45RA, CD201, CD90, ITGA3 and ZsG FACS intensities from (d) was calculated and is represented as 2d density plot of all cells (grey, n = 306,797). Cells falling into HSC- or ZsG-gates are overlayed and color-coded as in (d). g-h) HLF-reporter expression in immuno-phenotypic HSC gates. Reverse gating of the same data as above showing reporter expression in increasingly restricted HSC gates. Aggregated FACS data from all repeats in (g), summary in (h). i) Extrapolated HLF expression within immuno-phenotypic HSC gates. Ranges of cell fractions expressing HLF are summarized for each sub-population under consideration of measured allelic HR ratios of 20.2% (a). Lower boundaries are based on a scenario where all ZsG+ cells carry bi-allelic reporter integration, upper values are based on entirely mono-allelic integration.

Techniques Used: Expressing, Derivative Assay, Transduction, FACS, Ex Vivo

32) Product Images from "Chronic Inflammation-Related HPV: A Driving Force Speeds Oropharyngeal Carcinogenesis"

Article Title: Chronic Inflammation-Related HPV: A Driving Force Speeds Oropharyngeal Carcinogenesis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0133681

HPV infection correlates with the progression of oropharyngeal carcinogenesis. (A) Percentage of HPV 16 positive infection in patient tissues with normal oral mucosa, dysplasia, cancer in situ and cancer was shown. (B) Representative flow cytometry image of CD11b+ LIN- HLA-DR- CD33+ MDSCs in tissues of OPSCC patients with HPV-negative and HPV-positive. We first examined the percentage of LIN- HLA-DR- cells, and then screened the percentage of CD11b+ CD33+ cells in LIN- HLA-DR- cells. This image showed that the percentage of CD11b+ LIN- HLA-DR- CD33+ MDSCs in HPV-negative and HPV-positive OPSCC patients was 9.39% and 13.81%, respectively. (C) Representative immunohistochemical image (C3) of MPO in cancer tissues of OPSCC patients. C1 and C2 were H E staining and C2 was amplification of C1.
Figure Legend Snippet: HPV infection correlates with the progression of oropharyngeal carcinogenesis. (A) Percentage of HPV 16 positive infection in patient tissues with normal oral mucosa, dysplasia, cancer in situ and cancer was shown. (B) Representative flow cytometry image of CD11b+ LIN- HLA-DR- CD33+ MDSCs in tissues of OPSCC patients with HPV-negative and HPV-positive. We first examined the percentage of LIN- HLA-DR- cells, and then screened the percentage of CD11b+ CD33+ cells in LIN- HLA-DR- cells. This image showed that the percentage of CD11b+ LIN- HLA-DR- CD33+ MDSCs in HPV-negative and HPV-positive OPSCC patients was 9.39% and 13.81%, respectively. (C) Representative immunohistochemical image (C3) of MPO in cancer tissues of OPSCC patients. C1 and C2 were H E staining and C2 was amplification of C1.

Techniques Used: Infection, In Situ, Flow Cytometry, Cytometry, Immunohistochemistry, Staining, Amplification

33) Product Images from "Changes in Blood Components in Aphtha Patients with Excess Heat"

Article Title: Changes in Blood Components in Aphtha Patients with Excess Heat

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

doi: 10.1155/2016/7219257

Percentage of CD4 + CD25 + Treg cells in the peripheral blood lymphocytes from the NOR group and EXP group. (a) Flow cytometry of CD4 + CD25 + Treg cells from partial samples of the NOR group and EXP group. (b) Percentage of CD4 + CD25 + Treg cells in the peripheral blood lymphocytes from the NOR group and EXP group ( n = 20 per group). Note: NOR: normal group; EXP: experiment group. Comparing with the NOR group, ∗∗∗ P
Figure Legend Snippet: Percentage of CD4 + CD25 + Treg cells in the peripheral blood lymphocytes from the NOR group and EXP group. (a) Flow cytometry of CD4 + CD25 + Treg cells from partial samples of the NOR group and EXP group. (b) Percentage of CD4 + CD25 + Treg cells in the peripheral blood lymphocytes from the NOR group and EXP group ( n = 20 per group). Note: NOR: normal group; EXP: experiment group. Comparing with the NOR group, ∗∗∗ P

Techniques Used: Flow Cytometry, Cytometry

34) Product Images from "Impaired T-bet-pSTAT1α and perforin-mediated immune responses in the tumoral region of lung adenocarcinoma"

Article Title: Impaired T-bet-pSTAT1α and perforin-mediated immune responses in the tumoral region of lung adenocarcinoma

Journal: British Journal of Cancer

doi: 10.1038/bjc.2015.255

Reduced STAT1 phosphorylation in the tumoral region of NSCLC. ( A – F ) Western blot analysis of pSTAT1 and actin expression in lung tissue samples from the tumoral, peri-tumoral and control region of patients with adenocarcinoma (ADC) ( N Control =5; N Peri-tumoral =5, N Tumoral =5). Bar charts show mean values of the protein expression levels of pSTAT1 α -Isoform ( C and D ) and pSTAT1 β -Isoform ( E and F ) relative to actin levels in ADC and in SCC, respectively. ( G ) Flow cytometry analysis of pSTAT1 + cells in the tumoral, the peri-tumoral and the control lung region of one representative patient with NSCLC. pSTAT1 staining was performed with the lung cell suspensions after 20 min of incubation at 37 °C in the presence or absence of IFN- γ . pSTAT1 + cells were gated on CD4 + , CD8 + or CD11b + cells, respectively. ( H ) Flow cytometry analysis of pSTAT1 + CD4 + and pSTAT1 + CD8 + T cells gated on lymphocytes as well as pSTAT1 + CD11b + cells gated on big cells in the control, the peri-tumoral and the tumoral lung region of one representative patient with NSCLC. Data are shown as mean values±s.e.m. using Student's t -test * P =0.05.
Figure Legend Snippet: Reduced STAT1 phosphorylation in the tumoral region of NSCLC. ( A – F ) Western blot analysis of pSTAT1 and actin expression in lung tissue samples from the tumoral, peri-tumoral and control region of patients with adenocarcinoma (ADC) ( N Control =5; N Peri-tumoral =5, N Tumoral =5). Bar charts show mean values of the protein expression levels of pSTAT1 α -Isoform ( C and D ) and pSTAT1 β -Isoform ( E and F ) relative to actin levels in ADC and in SCC, respectively. ( G ) Flow cytometry analysis of pSTAT1 + cells in the tumoral, the peri-tumoral and the control lung region of one representative patient with NSCLC. pSTAT1 staining was performed with the lung cell suspensions after 20 min of incubation at 37 °C in the presence or absence of IFN- γ . pSTAT1 + cells were gated on CD4 + , CD8 + or CD11b + cells, respectively. ( H ) Flow cytometry analysis of pSTAT1 + CD4 + and pSTAT1 + CD8 + T cells gated on lymphocytes as well as pSTAT1 + CD11b + cells gated on big cells in the control, the peri-tumoral and the tumoral lung region of one representative patient with NSCLC. Data are shown as mean values±s.e.m. using Student's t -test * P =0.05.

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

Increased killing capability of CD8 + T cells from the control area. CD8 + T cells were isolated from the tumoral, peri-tumoral and control region of tissue samples from the lung of lung cancer patients. CD8 + cells were co-cultured with total cells from the tumoral region of the respective patient (tumour cells). Apoptosis and necrosis levels were evaluated by flow cytometry. Cells were double-stained with antibodies against Annexin V and PI. ( A ) Tumour cell gate and a representative flow cytometry plot of Annexin V versus PI staining is depicted. ( B – D ) Bar graphs show the mean percentages of Annexin V or PI single positive as well as Annexin V and PI double positive cells. Data are shown as mean values±s.e.m. using Student's t -test * P =0.05.
Figure Legend Snippet: Increased killing capability of CD8 + T cells from the control area. CD8 + T cells were isolated from the tumoral, peri-tumoral and control region of tissue samples from the lung of lung cancer patients. CD8 + cells were co-cultured with total cells from the tumoral region of the respective patient (tumour cells). Apoptosis and necrosis levels were evaluated by flow cytometry. Cells were double-stained with antibodies against Annexin V and PI. ( A ) Tumour cell gate and a representative flow cytometry plot of Annexin V versus PI staining is depicted. ( B – D ) Bar graphs show the mean percentages of Annexin V or PI single positive as well as Annexin V and PI double positive cells. Data are shown as mean values±s.e.m. using Student's t -test * P =0.05.

Techniques Used: Isolation, Cell Culture, Flow Cytometry, Cytometry, Staining

Reduced TBX21 and IFNG mRNA expression in the tumoral region of NSCLC. ( A and B ) qPCR-based expression analysis of TBX21 mRNA in lung tissue samples from the tumoral, peri-tumoral and control region of patients with adenocarcinoma (ADC) or squamous cell carcinoma (SCC; ADC: N Control =16, N Peri-tumoral =15, N Tumoral =16; SCC: N Control =17, N Peri-tumoral =17, N Tumoral =17). ( C and D ) Correlation between the tumour diameter and the TBX21 mRNA expression in the control area of patients with ADC and SCC. ( E and F ) qPCR-based expression analysis of IFNG mRNA in lung tissue samples from the tumoral, peri-tumoral and control region of patients with ADC or SCC (ADC: N Control =14, N Peri-tumoral =13, N Tumoral =14; SCC: N Control =16, N Peri-tumoral =16, N Tumoral =17). ( G ) Correlation between TBX21 and IFNG mRNA levels in the control as well as in the peri-tumoral and the tumoral area of patients with non-small-cell lung cancer (NSCLC). ( H ) Flow cytometry analysis of T-bet + CD4 + and T-bet + CD8 + T cells gated on lymphocytes from the control, the peri-tumoral and the tumoral lung region of one representative patient with NSCLC. Data are shown as mean values±s.e.m. using Student's t -test * P =0.05; ** P= 0.01; *** P= 0.001.
Figure Legend Snippet: Reduced TBX21 and IFNG mRNA expression in the tumoral region of NSCLC. ( A and B ) qPCR-based expression analysis of TBX21 mRNA in lung tissue samples from the tumoral, peri-tumoral and control region of patients with adenocarcinoma (ADC) or squamous cell carcinoma (SCC; ADC: N Control =16, N Peri-tumoral =15, N Tumoral =16; SCC: N Control =17, N Peri-tumoral =17, N Tumoral =17). ( C and D ) Correlation between the tumour diameter and the TBX21 mRNA expression in the control area of patients with ADC and SCC. ( E and F ) qPCR-based expression analysis of IFNG mRNA in lung tissue samples from the tumoral, peri-tumoral and control region of patients with ADC or SCC (ADC: N Control =14, N Peri-tumoral =13, N Tumoral =14; SCC: N Control =16, N Peri-tumoral =16, N Tumoral =17). ( G ) Correlation between TBX21 and IFNG mRNA levels in the control as well as in the peri-tumoral and the tumoral area of patients with non-small-cell lung cancer (NSCLC). ( H ) Flow cytometry analysis of T-bet + CD4 + and T-bet + CD8 + T cells gated on lymphocytes from the control, the peri-tumoral and the tumoral lung region of one representative patient with NSCLC. Data are shown as mean values±s.e.m. using Student's t -test * P =0.05; ** P= 0.01; *** P= 0.001.

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Flow Cytometry, Cytometry

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

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

Journal: PLoS ONE

doi: 10.1371/journal.pone.0162456

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

Techniques Used: Incubation, Western Blot

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

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

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

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

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

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

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

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

36) Product Images from "The ITIM-containing receptor LAIR1 is essential for acute myeloid leukemia development"

Article Title: The ITIM-containing receptor LAIR1 is essential for acute myeloid leukemia development

Journal: Nature cell biology

doi: 10.1038/ncb3158

Knockdown of lair1 blocks xenograft of human leukemia cell lines A, MV4-11 cells (1×10 6 cells) were infected with virus designed to express GFP and either scrambled shRNA or shRNA 226. GFP + cells were collected and transplanted into NSG mice (n = 7 mice) one day post-infection. Left panels show representative flow cytometry plots indicating decreased BM engraftment of MV4-11 cells treated with shRNA targeting lair1 . Staining with anti-human CD45 and anti-LAIR1 antibodies confirmed engraftment was from transplanted human leukemia cells. The percentages of each population are indicated in red numbers, and the median fluorescent intensity of LAIR1 expression is indicated in black numbers. The panel on the right plots percentages of GFP + cells in BM, spleen, liver, and PB at 1 month after transplantation (mean ± s.e.m., Student's t-test; n=7 samples, BM *** p
Figure Legend Snippet: Knockdown of lair1 blocks xenograft of human leukemia cell lines A, MV4-11 cells (1×10 6 cells) were infected with virus designed to express GFP and either scrambled shRNA or shRNA 226. GFP + cells were collected and transplanted into NSG mice (n = 7 mice) one day post-infection. Left panels show representative flow cytometry plots indicating decreased BM engraftment of MV4-11 cells treated with shRNA targeting lair1 . Staining with anti-human CD45 and anti-LAIR1 antibodies confirmed engraftment was from transplanted human leukemia cells. The percentages of each population are indicated in red numbers, and the median fluorescent intensity of LAIR1 expression is indicated in black numbers. The panel on the right plots percentages of GFP + cells in BM, spleen, liver, and PB at 1 month after transplantation (mean ± s.e.m., Student's t-test; n=7 samples, BM *** p

Techniques Used: Infection, shRNA, Mouse Assay, Flow Cytometry, Cytometry, Staining, Expressing, Transplantation Assay

37) Product Images from "cFLIP overexpression in T cells in thymoma-associated myasthenia gravis"

Article Title: cFLIP overexpression in T cells in thymoma-associated myasthenia gravis

Journal: Annals of Clinical and Translational Neurology

doi: 10.1002/acn3.210

Survival analysis of PBMCs from blood donors ( n = 16, age range 20–80 years) and thymoma patients ( n = 10; four MG(−) [two AB and two B2 subtype] and six TAMG(+) [three AB, two B2, and one B3 subtypes]) by MTT assay after treatment with FASLG (5 and 10 ng/mL for 24 h). There was a significant difference between thymoma patients and blood donors but not between TAMG(+) and MG(−) thymoma patients. Shown is one representative experiment (of two independent investigations; each assay was run in triplicate). PBMCs, peripheral blood mononuclear cells; TAMG, thymoma-associated myasthenia gravis; FASLG, Fas Ligand.
Figure Legend Snippet: Survival analysis of PBMCs from blood donors ( n = 16, age range 20–80 years) and thymoma patients ( n = 10; four MG(−) [two AB and two B2 subtype] and six TAMG(+) [three AB, two B2, and one B3 subtypes]) by MTT assay after treatment with FASLG (5 and 10 ng/mL for 24 h). There was a significant difference between thymoma patients and blood donors but not between TAMG(+) and MG(−) thymoma patients. Shown is one representative experiment (of two independent investigations; each assay was run in triplicate). PBMCs, peripheral blood mononuclear cells; TAMG, thymoma-associated myasthenia gravis; FASLG, Fas Ligand.

Techniques Used: MTT Assay

Impact of the NF- κ B inhibitor EF24 on survival of PBMCs from thymoma patients and blood donors. (A) Cell survival analysis using the MTT assay of PBMCs from nine thymoma patients (four with AB, two with B2, and three B3 subtypes) treated either with EF24 alone or with EF24 followed by FASLG). (B) Apoptosis measurement by AnnexinV/APC/PI flow cytometry (FACS, Fluorescence-Activated Cell Sorting Guawa Millipore) in PBMCs of four blood donors and four thymoma patients. PBMCs were treated either with FASLG (10 ng/mL) or with EF24 for 24 h followed by FASLG (10 ng/mL) for an additional 6 h. 25, 39, 54, 61 50, 58, 66, and 72 denote the ages of the patients (in years) from whom blood sample were obtained. PBMCs, peripheral blood mononuclear cells; FASLG, Fas Ligand.
Figure Legend Snippet: Impact of the NF- κ B inhibitor EF24 on survival of PBMCs from thymoma patients and blood donors. (A) Cell survival analysis using the MTT assay of PBMCs from nine thymoma patients (four with AB, two with B2, and three B3 subtypes) treated either with EF24 alone or with EF24 followed by FASLG). (B) Apoptosis measurement by AnnexinV/APC/PI flow cytometry (FACS, Fluorescence-Activated Cell Sorting Guawa Millipore) in PBMCs of four blood donors and four thymoma patients. PBMCs were treated either with FASLG (10 ng/mL) or with EF24 for 24 h followed by FASLG (10 ng/mL) for an additional 6 h. 25, 39, 54, 61 50, 58, 66, and 72 denote the ages of the patients (in years) from whom blood sample were obtained. PBMCs, peripheral blood mononuclear cells; FASLG, Fas Ligand.

Techniques Used: MTT Assay, Flow Cytometry, Cytometry, FACS, Fluorescence

38) Product Images from "CXCR7-mediated progression of osteosarcoma in the lungs"

Article Title: CXCR7-mediated progression of osteosarcoma in the lungs

Journal: British Journal of Cancer

doi: 10.1038/bjc.2013.482

Expression of CXCR7 and its ligands in murine and human tissues. ( A , B ) Immunohistochemical analysis of CXCR7 expression on human biopsies of primary skeletal OS ( A ) and lung metastatic lesions ( B ). The filled arrows show CXCR7-stained tumour-associated vessels, whereas the open arrow signals CXCR7-stained OS cells. Magnifications × 400. ( C ) Flow cytometry investigation of surface CXCR7 expression on K7M2 and SaOS-LM7 cells. Grey histograms represent cells stained with the isotype-matched control antibody. Black histograms show cells stained with an anti-CXCR7 antibody. Panels are representative of two independent experiments. ( D ) CXCR7 immunostaining of lung sections from SaOS-LM7-bearing mice (upper panel). Spleen sections from nude mouse are used as CXCR7-positive tissue control (lower panel). Positive staining is depicted as brown. Magnification × 200. ( E ) Quantitative real-time PCR for the analysis of CXCR7 ligands expression in mouse healthy lungs. The relative expression levels of genes were calculated using 18S as a normalising gene and expressed as 1/ΔC T. The mean±s.e.m. of duplicate per condition is shown. Representative of three independent experiments.
Figure Legend Snippet: Expression of CXCR7 and its ligands in murine and human tissues. ( A , B ) Immunohistochemical analysis of CXCR7 expression on human biopsies of primary skeletal OS ( A ) and lung metastatic lesions ( B ). The filled arrows show CXCR7-stained tumour-associated vessels, whereas the open arrow signals CXCR7-stained OS cells. Magnifications × 400. ( C ) Flow cytometry investigation of surface CXCR7 expression on K7M2 and SaOS-LM7 cells. Grey histograms represent cells stained with the isotype-matched control antibody. Black histograms show cells stained with an anti-CXCR7 antibody. Panels are representative of two independent experiments. ( D ) CXCR7 immunostaining of lung sections from SaOS-LM7-bearing mice (upper panel). Spleen sections from nude mouse are used as CXCR7-positive tissue control (lower panel). Positive staining is depicted as brown. Magnification × 200. ( E ) Quantitative real-time PCR for the analysis of CXCR7 ligands expression in mouse healthy lungs. The relative expression levels of genes were calculated using 18S as a normalising gene and expressed as 1/ΔC T. The mean±s.e.m. of duplicate per condition is shown. Representative of three independent experiments.

Techniques Used: Expressing, Immunohistochemistry, Staining, Flow Cytometry, Cytometry, Immunostaining, Mouse Assay, Real-time Polymerase Chain Reaction

39) Product Images from "Modulation of Human Immune Responses by Bovine Interleukin-10"

Article Title: Modulation of Human Immune Responses by Bovine Interleukin-10

Journal: PLoS ONE

doi: 10.1371/journal.pone.0018188

Bovine IL-10 dose-dependently inhibits DC surface marker expression and cytokine production. A: Typical example of raw data of flow cytometric analysis. Data shown is CD80 expression during LPS induced DC maturation of one donor. The solid line indicates CD80 staining and the dashed line the isotype control. On top of the graphs is indicated whether medium, LPS or LPS plus different doses of IL-10 (ng/ml) were used. B: Bovine IL-10 dose-dependently modulates DC surface marker expression (CD83, p = 0.002; CD40, p = 0.030; CD80, p = 0.018) during LPS induced maturation. Relative values are shown from three different donors. Mean fluorescent intensities were divided by the isotype control and expressed relative to the positive control (LPS, without IL-10), which was set at 100%. C: Recombinant bovine IL-10 dose dependently modulates the production of cytokines by human DC's during LPS induced maturation (IL-12, p =
Figure Legend Snippet: Bovine IL-10 dose-dependently inhibits DC surface marker expression and cytokine production. A: Typical example of raw data of flow cytometric analysis. Data shown is CD80 expression during LPS induced DC maturation of one donor. The solid line indicates CD80 staining and the dashed line the isotype control. On top of the graphs is indicated whether medium, LPS or LPS plus different doses of IL-10 (ng/ml) were used. B: Bovine IL-10 dose-dependently modulates DC surface marker expression (CD83, p = 0.002; CD40, p = 0.030; CD80, p = 0.018) during LPS induced maturation. Relative values are shown from three different donors. Mean fluorescent intensities were divided by the isotype control and expressed relative to the positive control (LPS, without IL-10), which was set at 100%. C: Recombinant bovine IL-10 dose dependently modulates the production of cytokines by human DC's during LPS induced maturation (IL-12, p =

Techniques Used: Marker, Expressing, Flow Cytometry, Staining, Positive Control, Recombinant

40) Product Images from "Modeling Initiation of Ewing Sarcoma in Human Neural Crest Cells"

Article Title: Modeling Initiation of Ewing Sarcoma in Human Neural Crest Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0019305

Flow cytometry confirms MSC markers in adherent hNCSC. hNCSC were isolated from in vitro differentiating hESC using p75-FACS. Nearly 50% of the cells also expressed the MSC marker CD73 on Day 0. After 2 days in adherent conditions the morphology of isolated cells changed from small cuboidal cells to larger mesenchymal cells (see Fig. 1A ). Consistent with this morphologic change flow cytometric analysis shows that after 5 days in culture most cells continue to express p75 but, in addition, nearly all cells express the MSC-associated markers CD73, CD105, CD90, CD29, and CD44.
Figure Legend Snippet: Flow cytometry confirms MSC markers in adherent hNCSC. hNCSC were isolated from in vitro differentiating hESC using p75-FACS. Nearly 50% of the cells also expressed the MSC marker CD73 on Day 0. After 2 days in adherent conditions the morphology of isolated cells changed from small cuboidal cells to larger mesenchymal cells (see Fig. 1A ). Consistent with this morphologic change flow cytometric analysis shows that after 5 days in culture most cells continue to express p75 but, in addition, nearly all cells express the MSC-associated markers CD73, CD105, CD90, CD29, and CD44.

Techniques Used: Flow Cytometry, Cytometry, Isolation, In Vitro, FACS, Marker

Related Articles

Centrifugation:

Article Title: Effective Non-Viral Delivery of siRNA to Acute Myeloid Leukemia Cells with Lipid-Substituted Polyethylenimines
Article Snippet: .. At day 2 and day 3, cells were stained with 4 µL of PE-labeled mouse anti-human CXCR4 (CD184) or PE-labeled mouse IgG isotype control (BD Pharmingen) antibody in 90 µL of medium (after centrifugation and resuspension) for 45 min at room temperature. .. They were subsequently re-suspended in HBSS and fixed in 3.7% formalin and immediately analysed by flow cytometry (FL2 channel).

Flow Cytometry:

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
Article Snippet: .. Flow cytometry PBMCs were incubated at 4°C for 15 minutes with the following fluorochrome-labeled anti-human monoclonal antibodies in PBS/0.2% BSA: anti-CD19-phycoerythrin-Cy7 (PE-Cy7, clone SJ25C1; BD Biosciences, San Jose, CA, USA), anti-CD3-Pacific Blue (PacB, clone UCHT1; BD Biosciences), anti-CD14-PacB (clone M5E2; BD Biosciences), anti-CD27-Cy5 (clone 2E4, kind gift from René van Lier, Academic Medical Center, University of Amsterdam, The Netherlands), anti-CD74-FITC (clone M-B741; BD Biosciences), milatuzumab-PE (hLL1; Immunomedics, Inc., Morris Plains, NJ, USA, labeled in-house at the Deutsches Rheumaforschungszentrum), anti-CD44-FITC (clone L178; BD Biosciences), anti-CXCR4-PE (clone 12G5; BD Biosciences), anti-β1-integrin-PE (clone MAR4; BD Biosciences), anti-CD62L-FITC (clone 145/15; Miltenyi Biotec GmbH, Bergisch Gladbach, Germany), and anti-CD9-FITC (clone MM2/57; Chemicon, Schwalbach, Germany). .. Rat anti-mouse anti-β7-integrin-PE (clone FIB504; BD Biosciences), which cross-reacts with human β7-integrin, was used to detect human β7-integrin, as described before [ - ].

Article Title: Extracellular Vesicles Released by Leishmania (Leishmania) amazonensis Promote Disease Progression and Induce the Production of Different Cytokines in Macrophages and B-1 Cells
Article Snippet: .. Flow cytometry measurements were performed on a BD FACS Accuri C6 flow cytometer (BD Biosciences) and the data evaluated by FCAP ArrayTM software (BD Bioscience). .. Cytokine standards were used to construct calibration curves, which were necessary for determining the cytokine concentrations in the test samples.

Article Title: Preexisting Virus-Specific T Lymphocytes-Mediated Enhancement of Adenovirus Infections to Human Blood CD14+ Cells
Article Snippet: .. For detecting the expression of EGFP reporter gene in different cell population, the infected PBMCs were incubated with corresponding fluorescent-labeled monoclonal antibodies (CD3-APC, CD3-PE, CD3-PerCP, CD14-APC, CD14-PE, CD19-PE-cy5, CD56-PE, CD27-APC, CD95-PE, HLADR-APC, Ki67-PE, 7-AAD, BD Pharmingen, San Diego, CA, USA) and CD38-FITC (STEMCELL Technologies, Vancouver, Canada), Integrinβ5-PE (eBioscience, San Diego, CA, USA), and then detected with a BD FACS LSR Fortessa flow cytometer (BD Biosciences, San Diego, CA, USA). .. For detecting the expression of SEAP reporter gene, PBMCs were seeded at 5 × 105 cells per well in 96-well plates, and then incubated with the indicated dosage of Ad-SEAP for 24–48 h at 37 °C in 5% CO2 incubator.

Labeling:

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
Article Snippet: .. Flow cytometry PBMCs were incubated at 4°C for 15 minutes with the following fluorochrome-labeled anti-human monoclonal antibodies in PBS/0.2% BSA: anti-CD19-phycoerythrin-Cy7 (PE-Cy7, clone SJ25C1; BD Biosciences, San Jose, CA, USA), anti-CD3-Pacific Blue (PacB, clone UCHT1; BD Biosciences), anti-CD14-PacB (clone M5E2; BD Biosciences), anti-CD27-Cy5 (clone 2E4, kind gift from René van Lier, Academic Medical Center, University of Amsterdam, The Netherlands), anti-CD74-FITC (clone M-B741; BD Biosciences), milatuzumab-PE (hLL1; Immunomedics, Inc., Morris Plains, NJ, USA, labeled in-house at the Deutsches Rheumaforschungszentrum), anti-CD44-FITC (clone L178; BD Biosciences), anti-CXCR4-PE (clone 12G5; BD Biosciences), anti-β1-integrin-PE (clone MAR4; BD Biosciences), anti-CD62L-FITC (clone 145/15; Miltenyi Biotec GmbH, Bergisch Gladbach, Germany), and anti-CD9-FITC (clone MM2/57; Chemicon, Schwalbach, Germany). .. Rat anti-mouse anti-β7-integrin-PE (clone FIB504; BD Biosciences), which cross-reacts with human β7-integrin, was used to detect human β7-integrin, as described before [ - ].

Cytometry:

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
Article Snippet: .. Flow cytometry PBMCs were incubated at 4°C for 15 minutes with the following fluorochrome-labeled anti-human monoclonal antibodies in PBS/0.2% BSA: anti-CD19-phycoerythrin-Cy7 (PE-Cy7, clone SJ25C1; BD Biosciences, San Jose, CA, USA), anti-CD3-Pacific Blue (PacB, clone UCHT1; BD Biosciences), anti-CD14-PacB (clone M5E2; BD Biosciences), anti-CD27-Cy5 (clone 2E4, kind gift from René van Lier, Academic Medical Center, University of Amsterdam, The Netherlands), anti-CD74-FITC (clone M-B741; BD Biosciences), milatuzumab-PE (hLL1; Immunomedics, Inc., Morris Plains, NJ, USA, labeled in-house at the Deutsches Rheumaforschungszentrum), anti-CD44-FITC (clone L178; BD Biosciences), anti-CXCR4-PE (clone 12G5; BD Biosciences), anti-β1-integrin-PE (clone MAR4; BD Biosciences), anti-CD62L-FITC (clone 145/15; Miltenyi Biotec GmbH, Bergisch Gladbach, Germany), and anti-CD9-FITC (clone MM2/57; Chemicon, Schwalbach, Germany). .. Rat anti-mouse anti-β7-integrin-PE (clone FIB504; BD Biosciences), which cross-reacts with human β7-integrin, was used to detect human β7-integrin, as described before [ - ].

Article Title: Extracellular Vesicles Released by Leishmania (Leishmania) amazonensis Promote Disease Progression and Induce the Production of Different Cytokines in Macrophages and B-1 Cells
Article Snippet: .. Flow cytometry measurements were performed on a BD FACS Accuri C6 flow cytometer (BD Biosciences) and the data evaluated by FCAP ArrayTM software (BD Bioscience). .. Cytokine standards were used to construct calibration curves, which were necessary for determining the cytokine concentrations in the test samples.

Article Title: Preexisting Virus-Specific T Lymphocytes-Mediated Enhancement of Adenovirus Infections to Human Blood CD14+ Cells
Article Snippet: .. For detecting the expression of EGFP reporter gene in different cell population, the infected PBMCs were incubated with corresponding fluorescent-labeled monoclonal antibodies (CD3-APC, CD3-PE, CD3-PerCP, CD14-APC, CD14-PE, CD19-PE-cy5, CD56-PE, CD27-APC, CD95-PE, HLADR-APC, Ki67-PE, 7-AAD, BD Pharmingen, San Diego, CA, USA) and CD38-FITC (STEMCELL Technologies, Vancouver, Canada), Integrinβ5-PE (eBioscience, San Diego, CA, USA), and then detected with a BD FACS LSR Fortessa flow cytometer (BD Biosciences, San Diego, CA, USA). .. For detecting the expression of SEAP reporter gene, PBMCs were seeded at 5 × 105 cells per well in 96-well plates, and then incubated with the indicated dosage of Ad-SEAP for 24–48 h at 37 °C in 5% CO2 incubator.

Incubation:

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
Article Snippet: .. Flow cytometry PBMCs were incubated at 4°C for 15 minutes with the following fluorochrome-labeled anti-human monoclonal antibodies in PBS/0.2% BSA: anti-CD19-phycoerythrin-Cy7 (PE-Cy7, clone SJ25C1; BD Biosciences, San Jose, CA, USA), anti-CD3-Pacific Blue (PacB, clone UCHT1; BD Biosciences), anti-CD14-PacB (clone M5E2; BD Biosciences), anti-CD27-Cy5 (clone 2E4, kind gift from René van Lier, Academic Medical Center, University of Amsterdam, The Netherlands), anti-CD74-FITC (clone M-B741; BD Biosciences), milatuzumab-PE (hLL1; Immunomedics, Inc., Morris Plains, NJ, USA, labeled in-house at the Deutsches Rheumaforschungszentrum), anti-CD44-FITC (clone L178; BD Biosciences), anti-CXCR4-PE (clone 12G5; BD Biosciences), anti-β1-integrin-PE (clone MAR4; BD Biosciences), anti-CD62L-FITC (clone 145/15; Miltenyi Biotec GmbH, Bergisch Gladbach, Germany), and anti-CD9-FITC (clone MM2/57; Chemicon, Schwalbach, Germany). .. Rat anti-mouse anti-β7-integrin-PE (clone FIB504; BD Biosciences), which cross-reacts with human β7-integrin, was used to detect human β7-integrin, as described before [ - ].

Article Title: Preexisting Virus-Specific T Lymphocytes-Mediated Enhancement of Adenovirus Infections to Human Blood CD14+ Cells
Article Snippet: .. For detecting the expression of EGFP reporter gene in different cell population, the infected PBMCs were incubated with corresponding fluorescent-labeled monoclonal antibodies (CD3-APC, CD3-PE, CD3-PerCP, CD14-APC, CD14-PE, CD19-PE-cy5, CD56-PE, CD27-APC, CD95-PE, HLADR-APC, Ki67-PE, 7-AAD, BD Pharmingen, San Diego, CA, USA) and CD38-FITC (STEMCELL Technologies, Vancouver, Canada), Integrinβ5-PE (eBioscience, San Diego, CA, USA), and then detected with a BD FACS LSR Fortessa flow cytometer (BD Biosciences, San Diego, CA, USA). .. For detecting the expression of SEAP reporter gene, PBMCs were seeded at 5 × 105 cells per well in 96-well plates, and then incubated with the indicated dosage of Ad-SEAP for 24–48 h at 37 °C in 5% CO2 incubator.

other:

Article Title: Effects of Pharmacological and Genetic Disruption of CXCR4 Chemokine Receptor Function in B-Cell Acute Lymphoblastic Leukaemia
Article Snippet: Anti-CXCR4-phycoerythrin (PE) antibodies (12G5 clone) were purchased from BD Pharmingen (San Jose, CA, USA) and anti-CXCR4-allophycocyanin (APC) antibodies (12G5 clone) were purchased from eBioscience (San Diego, CA, USA).

Infection:

Article Title: Preexisting Virus-Specific T Lymphocytes-Mediated Enhancement of Adenovirus Infections to Human Blood CD14+ Cells
Article Snippet: .. For detecting the expression of EGFP reporter gene in different cell population, the infected PBMCs were incubated with corresponding fluorescent-labeled monoclonal antibodies (CD3-APC, CD3-PE, CD3-PerCP, CD14-APC, CD14-PE, CD19-PE-cy5, CD56-PE, CD27-APC, CD95-PE, HLADR-APC, Ki67-PE, 7-AAD, BD Pharmingen, San Diego, CA, USA) and CD38-FITC (STEMCELL Technologies, Vancouver, Canada), Integrinβ5-PE (eBioscience, San Diego, CA, USA), and then detected with a BD FACS LSR Fortessa flow cytometer (BD Biosciences, San Diego, CA, USA). .. For detecting the expression of SEAP reporter gene, PBMCs were seeded at 5 × 105 cells per well in 96-well plates, and then incubated with the indicated dosage of Ad-SEAP for 24–48 h at 37 °C in 5% CO2 incubator.

Expressing:

Article Title: Preexisting Virus-Specific T Lymphocytes-Mediated Enhancement of Adenovirus Infections to Human Blood CD14+ Cells
Article Snippet: .. For detecting the expression of EGFP reporter gene in different cell population, the infected PBMCs were incubated with corresponding fluorescent-labeled monoclonal antibodies (CD3-APC, CD3-PE, CD3-PerCP, CD14-APC, CD14-PE, CD19-PE-cy5, CD56-PE, CD27-APC, CD95-PE, HLADR-APC, Ki67-PE, 7-AAD, BD Pharmingen, San Diego, CA, USA) and CD38-FITC (STEMCELL Technologies, Vancouver, Canada), Integrinβ5-PE (eBioscience, San Diego, CA, USA), and then detected with a BD FACS LSR Fortessa flow cytometer (BD Biosciences, San Diego, CA, USA). .. For detecting the expression of SEAP reporter gene, PBMCs were seeded at 5 × 105 cells per well in 96-well plates, and then incubated with the indicated dosage of Ad-SEAP for 24–48 h at 37 °C in 5% CO2 incubator.

Staining:

Article Title: Effective Non-Viral Delivery of siRNA to Acute Myeloid Leukemia Cells with Lipid-Substituted Polyethylenimines
Article Snippet: .. At day 2 and day 3, cells were stained with 4 µL of PE-labeled mouse anti-human CXCR4 (CD184) or PE-labeled mouse IgG isotype control (BD Pharmingen) antibody in 90 µL of medium (after centrifugation and resuspension) for 45 min at room temperature. .. They were subsequently re-suspended in HBSS and fixed in 3.7% formalin and immediately analysed by flow cytometry (FL2 channel).

Article Title: Circadian Expression of Migratory Factors Establishes Lineage-Specific Signatures that Guide the Homing of Leukocyte Subsets to Tissues
Article Snippet: .. Samples from mice engrafted with BS50 and C1498 leukemic cells were stained with anti-mouse CD45.1-PE-CF594 (clone A20, BD Biosciences) and anti-mouse CD45.2-AF700 (clone 104, Biolegend), while samples from NSG mice engrafted with NALM6 were stained with anti-human CD45-PE (BD PharMingen) and anti-mouse CD45-eFluor780 (clone 30-F11, eBiosciences). .. RBC were lysed using BD FACs Lysis buffer and samples were analyzed using a Fortessa (Becton Dickinson) flow cytometer.

FACS:

Article Title: Extracellular Vesicles Released by Leishmania (Leishmania) amazonensis Promote Disease Progression and Induce the Production of Different Cytokines in Macrophages and B-1 Cells
Article Snippet: .. Flow cytometry measurements were performed on a BD FACS Accuri C6 flow cytometer (BD Biosciences) and the data evaluated by FCAP ArrayTM software (BD Bioscience). .. Cytokine standards were used to construct calibration curves, which were necessary for determining the cytokine concentrations in the test samples.

Article Title: Preexisting Virus-Specific T Lymphocytes-Mediated Enhancement of Adenovirus Infections to Human Blood CD14+ Cells
Article Snippet: .. For detecting the expression of EGFP reporter gene in different cell population, the infected PBMCs were incubated with corresponding fluorescent-labeled monoclonal antibodies (CD3-APC, CD3-PE, CD3-PerCP, CD14-APC, CD14-PE, CD19-PE-cy5, CD56-PE, CD27-APC, CD95-PE, HLADR-APC, Ki67-PE, 7-AAD, BD Pharmingen, San Diego, CA, USA) and CD38-FITC (STEMCELL Technologies, Vancouver, Canada), Integrinβ5-PE (eBioscience, San Diego, CA, USA), and then detected with a BD FACS LSR Fortessa flow cytometer (BD Biosciences, San Diego, CA, USA). .. For detecting the expression of SEAP reporter gene, PBMCs were seeded at 5 × 105 cells per well in 96-well plates, and then incubated with the indicated dosage of Ad-SEAP for 24–48 h at 37 °C in 5% CO2 incubator.

Mouse Assay:

Article Title: Circadian Expression of Migratory Factors Establishes Lineage-Specific Signatures that Guide the Homing of Leukocyte Subsets to Tissues
Article Snippet: .. Samples from mice engrafted with BS50 and C1498 leukemic cells were stained with anti-mouse CD45.1-PE-CF594 (clone A20, BD Biosciences) and anti-mouse CD45.2-AF700 (clone 104, Biolegend), while samples from NSG mice engrafted with NALM6 were stained with anti-human CD45-PE (BD PharMingen) and anti-mouse CD45-eFluor780 (clone 30-F11, eBiosciences). .. RBC were lysed using BD FACs Lysis buffer and samples were analyzed using a Fortessa (Becton Dickinson) flow cytometer.

Article Title: Anti-leukemia efficacy and mechanisms of action of SL-101, a novel anti-CD123 antibody-conjugate, in acute myeloid leukemia
Article Snippet: .. The mice were sacrificed when they became moribund, and the spleen and BM were harvested to determine the extent of engraftment and to identify LSC phenotypes, defined as human CD45dim SSClow CD34+ CD38− CD123+ , using the antibodies CD45-APC-Cy7, CD38-PE-Cy7, CD123-PerCP-Cy5.5, CD34-APC (Cat. 555824; all from BD Biosciences). .. Additional PDX study was performed using AML11 injected into NSG-SGM3 (NSGS) mice (9–11-week old) breed in house (0.6 × 106 viable cells per mouse).

Software:

Article Title: Extracellular Vesicles Released by Leishmania (Leishmania) amazonensis Promote Disease Progression and Induce the Production of Different Cytokines in Macrophages and B-1 Cells
Article Snippet: .. Flow cytometry measurements were performed on a BD FACS Accuri C6 flow cytometer (BD Biosciences) and the data evaluated by FCAP ArrayTM software (BD Bioscience). .. Cytokine standards were used to construct calibration curves, which were necessary for determining the cytokine concentrations in the test samples.

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    Becton Dickinson anti cd8
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    Becton Dickinson flow cytometry allophycocyanin apc conjugated mouse anti human cd44 monoclonal antibody
    Effect of various concentrations of nicotine on <t>CD44</t> + CD24 − cell population. (A) Percentage of stem cells (CD44 + /CD24 − ) in the MCF-7 cell population was determined by flow <t>cytometry</t> analysis. (B) Expression patterns of CD24 and CD44 in MCF-7 cells were analyzed by flow cytometry for 24 and 48 h. Anti-CD24 antibody labeled with PE-Cy7 and anti-CD44 antibody labeled with <t>APC</t> were applied to the analysis. Gates are based on the isotype control corresponding to each cell line. PE-Cy7, phycoerythrin-cyanine 7; APC, <t>allophycocyanin;</t> CD, cluster of differentiation.
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    Radiation-induced lymphocyte apoptosis (RILA) flow cytometry profile Lymphocytes were selected (R1) from double-scatter dot plots (a-c) i.e. cell granularity (side scatter) and cell size (forward scatter). CD8 T-lymphocytes (R2) were then selected from R1 gated area as anti-CD4 or anti-CD8 FITC-conjugated antibody fixing cells (FL1H) (d-f) . CD4 (h) or CD8 (i) T-lymphocyte apoptosis rate (R3) was then calculated from the fraction of cells from R2 gated area with reduced size (forward scatter) and reduced DNA content i.e low propidium iodide labeling (FL2H). RILA was calculated after deduction of apoptosis rate of unirradiated controls (g) from irradiated samples (h, i) : respectively 20.1 and 27.5 % for CD4 and CD8 RILA in the present illustrative example.

    Journal: Oncotarget

    Article Title: Absence of correlation between radiation-induced CD8 T-lymphocyte apoptosis and sequelae in patients with prostate cancer accidentally overexposed to radiation

    doi: 10.18632/oncotarget.26001

    Figure Lengend Snippet: Radiation-induced lymphocyte apoptosis (RILA) flow cytometry profile Lymphocytes were selected (R1) from double-scatter dot plots (a-c) i.e. cell granularity (side scatter) and cell size (forward scatter). CD8 T-lymphocytes (R2) were then selected from R1 gated area as anti-CD4 or anti-CD8 FITC-conjugated antibody fixing cells (FL1H) (d-f) . CD4 (h) or CD8 (i) T-lymphocyte apoptosis rate (R3) was then calculated from the fraction of cells from R2 gated area with reduced size (forward scatter) and reduced DNA content i.e low propidium iodide labeling (FL2H). RILA was calculated after deduction of apoptosis rate of unirradiated controls (g) from irradiated samples (h, i) : respectively 20.1 and 27.5 % for CD4 and CD8 RILA in the present illustrative example.

    Article Snippet: CD4 and CD8 lymphocyte populations were selected using FITC-conjugated anti-CD4 (clone SK3) and anti-CD8 (clone SK1) mouse anti-human monoclonal antibodies, respectively (Becton Dickinson).

    Techniques: Flow Cytometry, Cytometry, Labeling, Irradiation

    Cumulative incidence of grade ≥2 digestive (left panels) or urinary (right panels) toxicity according to CD4 (top panels) and CD8 (bottom panels) RILA

    Journal: Oncotarget

    Article Title: Absence of correlation between radiation-induced CD8 T-lymphocyte apoptosis and sequelae in patients with prostate cancer accidentally overexposed to radiation

    doi: 10.18632/oncotarget.26001

    Figure Lengend Snippet: Cumulative incidence of grade ≥2 digestive (left panels) or urinary (right panels) toxicity according to CD4 (top panels) and CD8 (bottom panels) RILA

    Article Snippet: CD4 and CD8 lymphocyte populations were selected using FITC-conjugated anti-CD4 (clone SK3) and anti-CD8 (clone SK1) mouse anti-human monoclonal antibodies, respectively (Becton Dickinson).

    Techniques:

    CD69 expression in bone marrow B cells modulates the appearance of immature B cells in peripheral blood. (A) CD69 expression on bone marrow B cells. Results are shown as anti-CD69 fluorescence intensity for cells from control and B- S1pr1 KO mice compared with the isotype control. Representative results are from nine independent experiments. (B) Expression of the human CD69 transgene on bone marrow total B cells in control and transgenic (CD69-Tg) mice. B220 + bone marrow cells were analyzed for their expression of human CD69 by flow cytometry. (C and D) Distribution of B220 low IgM − (pro–/pre–) and B220 low IgM + (immature) B cells in the bone marrow (C) and of B220 + IgD low IgM + (immature) and B220 + IgD high IgM low (mature) B cells in the peripheral blood (D) of control and CD69 transgenic mice. Bars represent mean values, and the closed circles are individual mice. Data represent pooled results from three experiments. **, P

    Journal: The Journal of Experimental Medicine

    Article Title: S1P1 receptor directs the release of immature B cells from bone marrow into blood

    doi: 10.1084/jem.20092210

    Figure Lengend Snippet: CD69 expression in bone marrow B cells modulates the appearance of immature B cells in peripheral blood. (A) CD69 expression on bone marrow B cells. Results are shown as anti-CD69 fluorescence intensity for cells from control and B- S1pr1 KO mice compared with the isotype control. Representative results are from nine independent experiments. (B) Expression of the human CD69 transgene on bone marrow total B cells in control and transgenic (CD69-Tg) mice. B220 + bone marrow cells were analyzed for their expression of human CD69 by flow cytometry. (C and D) Distribution of B220 low IgM − (pro–/pre–) and B220 low IgM + (immature) B cells in the bone marrow (C) and of B220 + IgD low IgM + (immature) and B220 + IgD high IgM low (mature) B cells in the peripheral blood (D) of control and CD69 transgenic mice. Bars represent mean values, and the closed circles are individual mice. Data represent pooled results from three experiments. **, P

    Article Snippet: Offspring that were positive for the human CD69 and Cre transgenes were tested for the expression of the human CD69 protein on B cells by flow cytometry using an FITC-conjugated anti–human CD69 antibody (BD).

    Techniques: Expressing, Fluorescence, Mouse Assay, Transgenic Assay, Flow Cytometry, Cytometry

    Effect of various concentrations of nicotine on CD44 + CD24 − cell population. (A) Percentage of stem cells (CD44 + /CD24 − ) in the MCF-7 cell population was determined by flow cytometry analysis. (B) Expression patterns of CD24 and CD44 in MCF-7 cells were analyzed by flow cytometry for 24 and 48 h. Anti-CD24 antibody labeled with PE-Cy7 and anti-CD44 antibody labeled with APC were applied to the analysis. Gates are based on the isotype control corresponding to each cell line. PE-Cy7, phycoerythrin-cyanine 7; APC, allophycocyanin; CD, cluster of differentiation.

    Journal: Experimental and Therapeutic Medicine

    Article Title: Nicotine reduces effectiveness of doxorubicin chemotherapy and promotes CD44+CD24− cancer stem cells in MCF-7 cell populations

    doi: 10.3892/etm.2018.6149

    Figure Lengend Snippet: Effect of various concentrations of nicotine on CD44 + CD24 − cell population. (A) Percentage of stem cells (CD44 + /CD24 − ) in the MCF-7 cell population was determined by flow cytometry analysis. (B) Expression patterns of CD24 and CD44 in MCF-7 cells were analyzed by flow cytometry for 24 and 48 h. Anti-CD24 antibody labeled with PE-Cy7 and anti-CD44 antibody labeled with APC were applied to the analysis. Gates are based on the isotype control corresponding to each cell line. PE-Cy7, phycoerythrin-cyanine 7; APC, allophycocyanin; CD, cluster of differentiation.

    Article Snippet: CSC analysis by flow cytometry Allophycocyanin (APC)-conjugated mouse anti-human CD44 monoclonal antibody (cat. no. BD 559942) and phycoerythrin/cyanine 7 (PE/Cy7)-conjugated mouse anti-human CD24 monoclonal antibody (cat. no. BD 561646) were purchased from BD Pharmingen (BD Biosciences, Franklin Lakes, NJ, USA).

    Techniques: Flow Cytometry, Cytometry, Expressing, Labeling

    Impaired cell cycle progression of CD4+ and CD8+ T cells in mice exposed to METH. Splenocytes were stimulated with mouse anti-CD3/CD28 for 48 h to induce proliferation. After activation cells were stained with 7AAD and cell cycle data analysis was performed on CD4+ and CD8+ population. FlowJo Dean-Jett-Fox (DJF) cell cycle modeling algorithm was used to define the G1, S and G2 phases of the cell cycle. Results are representative of two different experiments from three mice per day. a Average values of % fraction of cells in respective phase in total gated CD4+ T-cell population. b Average values of % fraction of cells in respective phase in total gated CD8+ T-cell population. * P value

    Journal: Cell Death Discovery

    Article Title: Methamphetamine alters T cell cycle entry and progression: role in immune dysfunction

    doi: 10.1038/s41420-018-0045-6

    Figure Lengend Snippet: Impaired cell cycle progression of CD4+ and CD8+ T cells in mice exposed to METH. Splenocytes were stimulated with mouse anti-CD3/CD28 for 48 h to induce proliferation. After activation cells were stained with 7AAD and cell cycle data analysis was performed on CD4+ and CD8+ population. FlowJo Dean-Jett-Fox (DJF) cell cycle modeling algorithm was used to define the G1, S and G2 phases of the cell cycle. Results are representative of two different experiments from three mice per day. a Average values of % fraction of cells in respective phase in total gated CD4+ T-cell population. b Average values of % fraction of cells in respective phase in total gated CD8+ T-cell population. * P value

    Article Snippet: V500 rat anti-mouse CD4 and mouse anti-human CD8 antibody was purchased from Becton–Dickinson (Mountain View, CA).

    Techniques: Mouse Assay, Activation Assay, Staining

    Gating strategies for the flow cytometry analyses of peripheral blood leukocytes. Peripheral blood leukocytes from healthy individuals ( n = 5) were separated into two broad subsets following staining with cell surface marker CD20, using flow cytometry. Based on their distribution in a side scatter (SS) vs. forward scatter (FS) contour plot, viable leukocytes were selected. B lymphocytes were identified based on their binding of the CD20 antibody, while CD20-negative lymphocytes were identified as T lymphocytes. From this CD20-negative T lymphocytes population, CD4+ and CD8+ T cells were identified based on their binding to CD4 and CD8 antibodies, respectively

    Journal: Cell Death Discovery

    Article Title: Methamphetamine alters T cell cycle entry and progression: role in immune dysfunction

    doi: 10.1038/s41420-018-0045-6

    Figure Lengend Snippet: Gating strategies for the flow cytometry analyses of peripheral blood leukocytes. Peripheral blood leukocytes from healthy individuals ( n = 5) were separated into two broad subsets following staining with cell surface marker CD20, using flow cytometry. Based on their distribution in a side scatter (SS) vs. forward scatter (FS) contour plot, viable leukocytes were selected. B lymphocytes were identified based on their binding of the CD20 antibody, while CD20-negative lymphocytes were identified as T lymphocytes. From this CD20-negative T lymphocytes population, CD4+ and CD8+ T cells were identified based on their binding to CD4 and CD8 antibodies, respectively

    Article Snippet: V500 rat anti-mouse CD4 and mouse anti-human CD8 antibody was purchased from Becton–Dickinson (Mountain View, CA).

    Techniques: Flow Cytometry, Cytometry, Staining, Marker, Binding Assay

    METH alters cell cycle progression of CD4+ / CD8+ T cells. PBMCs were stimulated with human anti-CD3/CD28 for 48 h to induce proliferation. After activation, PBMCs were either serum starved or treated with METH (100 μM), Aphidicoline (100 ng/ml) or Nocodazole (25 μM) for 24 h. The cells were stained with 7AAD and cell cycle data analysis was performed on CD4+ and CD8+ population. FlowJo Dean-Jett-Fox (DJF) cell cycle modeling algorithm was used to define the G1, S and G2 phases of the cell cycle. The data are presented as cellular DNA content frequency histograms. The green overlay indicates the fit curves to the stages of the cell cycle. Results are representative of nine independent experiments from five separate donors. a Representative DNA content frequency histograms of CD4+ T cells. b The data from the cell cycle distribution of CD4+ T cells are summarized and presented as the mean ± SEM of nine independent experiments. c Representative DNA content frequency histograms of CD8+ T cells. d The data from the cell cycle distribution of CD8+ T cells are summarized and presented as the mean ± SEM of nine independent experiments. ∇ P

    Journal: Cell Death Discovery

    Article Title: Methamphetamine alters T cell cycle entry and progression: role in immune dysfunction

    doi: 10.1038/s41420-018-0045-6

    Figure Lengend Snippet: METH alters cell cycle progression of CD4+ / CD8+ T cells. PBMCs were stimulated with human anti-CD3/CD28 for 48 h to induce proliferation. After activation, PBMCs were either serum starved or treated with METH (100 μM), Aphidicoline (100 ng/ml) or Nocodazole (25 μM) for 24 h. The cells were stained with 7AAD and cell cycle data analysis was performed on CD4+ and CD8+ population. FlowJo Dean-Jett-Fox (DJF) cell cycle modeling algorithm was used to define the G1, S and G2 phases of the cell cycle. The data are presented as cellular DNA content frequency histograms. The green overlay indicates the fit curves to the stages of the cell cycle. Results are representative of nine independent experiments from five separate donors. a Representative DNA content frequency histograms of CD4+ T cells. b The data from the cell cycle distribution of CD4+ T cells are summarized and presented as the mean ± SEM of nine independent experiments. c Representative DNA content frequency histograms of CD8+ T cells. d The data from the cell cycle distribution of CD8+ T cells are summarized and presented as the mean ± SEM of nine independent experiments. ∇ P

    Article Snippet: V500 rat anti-mouse CD4 and mouse anti-human CD8 antibody was purchased from Becton–Dickinson (Mountain View, CA).

    Techniques: Activation Assay, Staining

    Suppressed cyclin E expression of CD4/CD8+ T cells after METH treatment. PBMCs were stimulated with human anti-CD3/CD28 for 48 h and then treated with METH (100 μM) for 24 h. a Representative histogram of fluorescence intensity of CD4+ T cells. b Fold change of Cyclin E expression in METH treated cells in respective phase in total gated CD4+ T-cell population. c Representative histogram of fluorescence intensity of CD8+ T cells. d Fold change of Cyclin E expression in METH treated cells in respective phase in total gated CD8+ T-cell population. The data from the cyclin E expression were summarized and presented as the mean ± SEM of five independent experiments. ∇ P

    Journal: Cell Death Discovery

    Article Title: Methamphetamine alters T cell cycle entry and progression: role in immune dysfunction

    doi: 10.1038/s41420-018-0045-6

    Figure Lengend Snippet: Suppressed cyclin E expression of CD4/CD8+ T cells after METH treatment. PBMCs were stimulated with human anti-CD3/CD28 for 48 h and then treated with METH (100 μM) for 24 h. a Representative histogram of fluorescence intensity of CD4+ T cells. b Fold change of Cyclin E expression in METH treated cells in respective phase in total gated CD4+ T-cell population. c Representative histogram of fluorescence intensity of CD8+ T cells. d Fold change of Cyclin E expression in METH treated cells in respective phase in total gated CD8+ T-cell population. The data from the cyclin E expression were summarized and presented as the mean ± SEM of five independent experiments. ∇ P

    Article Snippet: V500 rat anti-mouse CD4 and mouse anti-human CD8 antibody was purchased from Becton–Dickinson (Mountain View, CA).

    Techniques: Expressing, Fluorescence

    Impaired CDK2 expression of CD4/CD8+ T cells following METH treatment. PBMCs were exposed to METH (100 μM) for 24 h following stimulation with human anti-CD3/CD28 for 48 h. a Representative histogram of fluorescence intensity of CD4+ T cells. b Fold change of CDK2 expression in METH treated cells in respective phase in total gated CD4+ T-cell population. c Representative histogram of fluorescence intensity of CD8+ T cells. d Fold change of CDK2 expression in METH treated cells in respective phase in total gated CD8+ T-cell population. The data from the CDK2 expression were summarized and presented as the mean ± SEM of six independent experiments. ∇ P

    Journal: Cell Death Discovery

    Article Title: Methamphetamine alters T cell cycle entry and progression: role in immune dysfunction

    doi: 10.1038/s41420-018-0045-6

    Figure Lengend Snippet: Impaired CDK2 expression of CD4/CD8+ T cells following METH treatment. PBMCs were exposed to METH (100 μM) for 24 h following stimulation with human anti-CD3/CD28 for 48 h. a Representative histogram of fluorescence intensity of CD4+ T cells. b Fold change of CDK2 expression in METH treated cells in respective phase in total gated CD4+ T-cell population. c Representative histogram of fluorescence intensity of CD8+ T cells. d Fold change of CDK2 expression in METH treated cells in respective phase in total gated CD8+ T-cell population. The data from the CDK2 expression were summarized and presented as the mean ± SEM of six independent experiments. ∇ P

    Article Snippet: V500 rat anti-mouse CD4 and mouse anti-human CD8 antibody was purchased from Becton–Dickinson (Mountain View, CA).

    Techniques: Expressing, Fluorescence