monensin  (BioLegend)

 
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    Name:
    Monensin Solution 1 000X
    Description:
    Monensin Solution 1 000X Apps ICFC Size 1 ml
    Catalog Number:
    420701
    Price:
    45
    Category:
    Buffer Solution Chemical
    Applications:
    ICFC
    Size:
    1 ml
    Quantity:
    1
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    Structured Review

    BioLegend monensin
    Induction of differentiation, inhibition of cell growth, and resistance to NK cell-mediated cytotoxicity in OSCSCs induced by IL-2 + anti-CD16 mAb-treated and paraformaldehyde-fixed NK cells is mediated by the combination of IFN-γ and TNF-α and not each cytokine alone . Highly purified NK cells were left untreated or treated with the combination of IL-2 (1000 units/ml) and anti-CD16 mAb (3 μg/ml) for 24 h, after which the NK cells were fixed with 2% paraformaldehyde and added to OSCSCs (0.75:1 NK:OSCSC ratio) in the presence and absence of anti-TNF-α (1:100) and/or anti-IFN-γ (1:100) or isotype control antibodies for a period of 5 days. The media containing the fixed NK cells were removed and extensively washed from each treated OSCSCs and the cytotoxicity against OSCSCs were assessed using freshly isolated untreated NK cells or those treated with IL-2 using a standard 4 h 51 Cr release assay. The complete removal of fixed NK cells from OSCSCs was determined by microscopy. Percent cytotoxicity was obtained at different effector to target ratio, and the lytic units 30/10 6 cells were determined using inverse number of NK cells required to lyse 30% of the tumor cells × 100 (A) . The lack of cytotoxic function of 2% paraformaldehyde-fixed untreated and IL-2-treated NK cells were assessed against OSCSCs and OSCCs. NK cells were left untreated or treated with IL-2 for 24 h before they were fixed with 2% paraformaldehyde (B) . NK cells were purified from healthy donor and left untreated or treated with IL-2 (1000 units/ml) and the combination of IL-2 (1000 units/ml) and anti-CD16 mAb (3 μg/ml). After an overnight treatment, NK cells were fixed with freshly prepared 2% paraformaldehyde and washed twice with 1× PBS. After 24 h post fixation, supernatants were then collected and the levels of IFN-γ were measured with specific ELISA (C) . NK cells purified from healthy donors were left untreated or treated with a combination of IL-2 (1000 units/ml) and anti-CD16 mAb (3 μg/ml) for 24 h, after which the NK cells were washed extensively and stained with PE-conjugated anti-TNF-α mAb followed by flow cytometric analysis. IFN-γ expression was assessed using purified mouse anti-human IFN-γ antibody followed by PE-conjugated goat anti-mouse IgG. Isotype control antibodies were used as controls. The numbers on the right hand corner are percentage positive for each histogram. The histogram overlay for the anti-TNF-α or anti-IFN-γ stained untreated (left) and IL-2 + anti-CD16 mAb-treated NK cells (right) are also shown in this figure. (D) The surface expression of CD54, CD44, B7H1, and MHC class I on untreated and fixed NK-treated OSCSCs as described above were assessed after PE-conjugated antibody staining followed by flow cytometric analysis. Isotype control antibodies were used as controls. The numbers on the right hand corner are the percentages and the mean channel fluorescence intensities for each histogram (E) . At the end of the incubat ion of OSCSCs with fixed NK cells, OSCSCs which were remained attached to the plate were collected, and the number of cells (F) and their viability (G) were assessed using microscopy, and propidium iodide staining followed by flow cytometric analysis, respectively. Highly purified NK cells were left untreated or treated with the combination of IL-2 (1000 units/ml) and anti-CD16 mAb (3 μg/ml) in the presence and absence of <t>monensin</t> (1:1300) for 24 h, after which each sample of NK cells were extensively washed and fixed with 2% paraformaldehyde and added to OSCSCs (0.75:1 NK:OSCSC) for a period of 5 days. Inhibition of IFN-γ release in monensin-treated NK cells before fixation with 2% paraformaldehyde were assessed by ELISA (H) . The results were compared to OSCSCs in the absence of fixed NK cells. The media containing the fixed NK cells were removed and extensively washed from each treated OSCSCs and the cytotoxicity against each condition of OSCSCs were assessed using freshly isolated untreated NK cells (I) , those treated with IL-2 (J) or treated with the combination of IL-2 + anti-CD16 mAb (K) using a standard 4 h 51 Cr release assay. The complete removal of fixed NK cells from OSCSCs was determined by microscopy. Differentiated OSCCs were used as control for the differentiation of OSCSCs. Percent cytotoxicity was obtained at different effector to target ratio, and the lytic units 30/10 6 cells were determined using inverse number of NK cells required to lyse 30% of the tumor cells × 100. The surface expression of CD54, CD44, and MHC class I on untreated and fixed NK-treated OSCSCs as described above were assessed after PE-conjugated antibody staining followed by flow cytometric analysis. Isotype control antibodies were used as controls. The numbers on the right hand corner are the percentages and the mean channel fluorescence intensities for each histogram (L) . At the end of the incubation of OSCSCs with fixed NK cells, the media containing the fixed NK samples were removed from OSCSCs and the viability of the cells were assessed using propidium iodide staining followed by flow cytometric analysis (M) . One of minimum three representative experiments is shown in each of (A–M) .
    Monensin Solution 1 000X Apps ICFC Size 1 ml
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    Images

    1) Product Images from "Induction of Split Anergy Conditions Natural Killer Cells to Promote Differentiation of Stem Cells through Cell-Cell Contact and Secreted Factors"

    Article Title: Induction of Split Anergy Conditions Natural Killer Cells to Promote Differentiation of Stem Cells through Cell-Cell Contact and Secreted Factors

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2014.00269

    Induction of differentiation, inhibition of cell growth, and resistance to NK cell-mediated cytotoxicity in OSCSCs induced by IL-2 + anti-CD16 mAb-treated and paraformaldehyde-fixed NK cells is mediated by the combination of IFN-γ and TNF-α and not each cytokine alone . Highly purified NK cells were left untreated or treated with the combination of IL-2 (1000 units/ml) and anti-CD16 mAb (3 μg/ml) for 24 h, after which the NK cells were fixed with 2% paraformaldehyde and added to OSCSCs (0.75:1 NK:OSCSC ratio) in the presence and absence of anti-TNF-α (1:100) and/or anti-IFN-γ (1:100) or isotype control antibodies for a period of 5 days. The media containing the fixed NK cells were removed and extensively washed from each treated OSCSCs and the cytotoxicity against OSCSCs were assessed using freshly isolated untreated NK cells or those treated with IL-2 using a standard 4 h 51 Cr release assay. The complete removal of fixed NK cells from OSCSCs was determined by microscopy. Percent cytotoxicity was obtained at different effector to target ratio, and the lytic units 30/10 6 cells were determined using inverse number of NK cells required to lyse 30% of the tumor cells × 100 (A) . The lack of cytotoxic function of 2% paraformaldehyde-fixed untreated and IL-2-treated NK cells were assessed against OSCSCs and OSCCs. NK cells were left untreated or treated with IL-2 for 24 h before they were fixed with 2% paraformaldehyde (B) . NK cells were purified from healthy donor and left untreated or treated with IL-2 (1000 units/ml) and the combination of IL-2 (1000 units/ml) and anti-CD16 mAb (3 μg/ml). After an overnight treatment, NK cells were fixed with freshly prepared 2% paraformaldehyde and washed twice with 1× PBS. After 24 h post fixation, supernatants were then collected and the levels of IFN-γ were measured with specific ELISA (C) . NK cells purified from healthy donors were left untreated or treated with a combination of IL-2 (1000 units/ml) and anti-CD16 mAb (3 μg/ml) for 24 h, after which the NK cells were washed extensively and stained with PE-conjugated anti-TNF-α mAb followed by flow cytometric analysis. IFN-γ expression was assessed using purified mouse anti-human IFN-γ antibody followed by PE-conjugated goat anti-mouse IgG. Isotype control antibodies were used as controls. The numbers on the right hand corner are percentage positive for each histogram. The histogram overlay for the anti-TNF-α or anti-IFN-γ stained untreated (left) and IL-2 + anti-CD16 mAb-treated NK cells (right) are also shown in this figure. (D) The surface expression of CD54, CD44, B7H1, and MHC class I on untreated and fixed NK-treated OSCSCs as described above were assessed after PE-conjugated antibody staining followed by flow cytometric analysis. Isotype control antibodies were used as controls. The numbers on the right hand corner are the percentages and the mean channel fluorescence intensities for each histogram (E) . At the end of the incubat ion of OSCSCs with fixed NK cells, OSCSCs which were remained attached to the plate were collected, and the number of cells (F) and their viability (G) were assessed using microscopy, and propidium iodide staining followed by flow cytometric analysis, respectively. Highly purified NK cells were left untreated or treated with the combination of IL-2 (1000 units/ml) and anti-CD16 mAb (3 μg/ml) in the presence and absence of monensin (1:1300) for 24 h, after which each sample of NK cells were extensively washed and fixed with 2% paraformaldehyde and added to OSCSCs (0.75:1 NK:OSCSC) for a period of 5 days. Inhibition of IFN-γ release in monensin-treated NK cells before fixation with 2% paraformaldehyde were assessed by ELISA (H) . The results were compared to OSCSCs in the absence of fixed NK cells. The media containing the fixed NK cells were removed and extensively washed from each treated OSCSCs and the cytotoxicity against each condition of OSCSCs were assessed using freshly isolated untreated NK cells (I) , those treated with IL-2 (J) or treated with the combination of IL-2 + anti-CD16 mAb (K) using a standard 4 h 51 Cr release assay. The complete removal of fixed NK cells from OSCSCs was determined by microscopy. Differentiated OSCCs were used as control for the differentiation of OSCSCs. Percent cytotoxicity was obtained at different effector to target ratio, and the lytic units 30/10 6 cells were determined using inverse number of NK cells required to lyse 30% of the tumor cells × 100. The surface expression of CD54, CD44, and MHC class I on untreated and fixed NK-treated OSCSCs as described above were assessed after PE-conjugated antibody staining followed by flow cytometric analysis. Isotype control antibodies were used as controls. The numbers on the right hand corner are the percentages and the mean channel fluorescence intensities for each histogram (L) . At the end of the incubation of OSCSCs with fixed NK cells, the media containing the fixed NK samples were removed from OSCSCs and the viability of the cells were assessed using propidium iodide staining followed by flow cytometric analysis (M) . One of minimum three representative experiments is shown in each of (A–M) .
    Figure Legend Snippet: Induction of differentiation, inhibition of cell growth, and resistance to NK cell-mediated cytotoxicity in OSCSCs induced by IL-2 + anti-CD16 mAb-treated and paraformaldehyde-fixed NK cells is mediated by the combination of IFN-γ and TNF-α and not each cytokine alone . Highly purified NK cells were left untreated or treated with the combination of IL-2 (1000 units/ml) and anti-CD16 mAb (3 μg/ml) for 24 h, after which the NK cells were fixed with 2% paraformaldehyde and added to OSCSCs (0.75:1 NK:OSCSC ratio) in the presence and absence of anti-TNF-α (1:100) and/or anti-IFN-γ (1:100) or isotype control antibodies for a period of 5 days. The media containing the fixed NK cells were removed and extensively washed from each treated OSCSCs and the cytotoxicity against OSCSCs were assessed using freshly isolated untreated NK cells or those treated with IL-2 using a standard 4 h 51 Cr release assay. The complete removal of fixed NK cells from OSCSCs was determined by microscopy. Percent cytotoxicity was obtained at different effector to target ratio, and the lytic units 30/10 6 cells were determined using inverse number of NK cells required to lyse 30% of the tumor cells × 100 (A) . The lack of cytotoxic function of 2% paraformaldehyde-fixed untreated and IL-2-treated NK cells were assessed against OSCSCs and OSCCs. NK cells were left untreated or treated with IL-2 for 24 h before they were fixed with 2% paraformaldehyde (B) . NK cells were purified from healthy donor and left untreated or treated with IL-2 (1000 units/ml) and the combination of IL-2 (1000 units/ml) and anti-CD16 mAb (3 μg/ml). After an overnight treatment, NK cells were fixed with freshly prepared 2% paraformaldehyde and washed twice with 1× PBS. After 24 h post fixation, supernatants were then collected and the levels of IFN-γ were measured with specific ELISA (C) . NK cells purified from healthy donors were left untreated or treated with a combination of IL-2 (1000 units/ml) and anti-CD16 mAb (3 μg/ml) for 24 h, after which the NK cells were washed extensively and stained with PE-conjugated anti-TNF-α mAb followed by flow cytometric analysis. IFN-γ expression was assessed using purified mouse anti-human IFN-γ antibody followed by PE-conjugated goat anti-mouse IgG. Isotype control antibodies were used as controls. The numbers on the right hand corner are percentage positive for each histogram. The histogram overlay for the anti-TNF-α or anti-IFN-γ stained untreated (left) and IL-2 + anti-CD16 mAb-treated NK cells (right) are also shown in this figure. (D) The surface expression of CD54, CD44, B7H1, and MHC class I on untreated and fixed NK-treated OSCSCs as described above were assessed after PE-conjugated antibody staining followed by flow cytometric analysis. Isotype control antibodies were used as controls. The numbers on the right hand corner are the percentages and the mean channel fluorescence intensities for each histogram (E) . At the end of the incubat ion of OSCSCs with fixed NK cells, OSCSCs which were remained attached to the plate were collected, and the number of cells (F) and their viability (G) were assessed using microscopy, and propidium iodide staining followed by flow cytometric analysis, respectively. Highly purified NK cells were left untreated or treated with the combination of IL-2 (1000 units/ml) and anti-CD16 mAb (3 μg/ml) in the presence and absence of monensin (1:1300) for 24 h, after which each sample of NK cells were extensively washed and fixed with 2% paraformaldehyde and added to OSCSCs (0.75:1 NK:OSCSC) for a period of 5 days. Inhibition of IFN-γ release in monensin-treated NK cells before fixation with 2% paraformaldehyde were assessed by ELISA (H) . The results were compared to OSCSCs in the absence of fixed NK cells. The media containing the fixed NK cells were removed and extensively washed from each treated OSCSCs and the cytotoxicity against each condition of OSCSCs were assessed using freshly isolated untreated NK cells (I) , those treated with IL-2 (J) or treated with the combination of IL-2 + anti-CD16 mAb (K) using a standard 4 h 51 Cr release assay. The complete removal of fixed NK cells from OSCSCs was determined by microscopy. Differentiated OSCCs were used as control for the differentiation of OSCSCs. Percent cytotoxicity was obtained at different effector to target ratio, and the lytic units 30/10 6 cells were determined using inverse number of NK cells required to lyse 30% of the tumor cells × 100. The surface expression of CD54, CD44, and MHC class I on untreated and fixed NK-treated OSCSCs as described above were assessed after PE-conjugated antibody staining followed by flow cytometric analysis. Isotype control antibodies were used as controls. The numbers on the right hand corner are the percentages and the mean channel fluorescence intensities for each histogram (L) . At the end of the incubation of OSCSCs with fixed NK cells, the media containing the fixed NK samples were removed from OSCSCs and the viability of the cells were assessed using propidium iodide staining followed by flow cytometric analysis (M) . One of minimum three representative experiments is shown in each of (A–M) .

    Techniques Used: Inhibition, Purification, Isolation, Release Assay, Microscopy, Enzyme-linked Immunosorbent Assay, Staining, Flow Cytometry, Expressing, Fluorescence, Incubation

    2) Product Images from "Human intestinal tissue-resident memory T cells comprise transcriptionally and functionally distinct subsets"

    Article Title: Human intestinal tissue-resident memory T cells comprise transcriptionally and functionally distinct subsets

    Journal: Cell Reports

    doi: 10.1016/j.celrep.2020.108661

    CD103 + and CD103 − CD4 + T cells display analogous phenotypic and functional differences to their CD8 + counterparts (A) MFI (CD161, CD7, CD127, β2-integrin, granzyme K, and KLRG1) or percentage positive (Ki-67) of CD4 + T cells, categorized by CD69 and CD103 expression, in small intestinal biopsies from healthy control subjects (n = 10). MFI represented by bars. Connecting lines represent populations from the same subject. Blue, CD69 − CD103 − cells; red, CD69 + CD103 − cells; black, CD69 + CD103 + cells. (B) MFI of β2-integrin on recipient-derived CD4 + T cells, categorized by CD69 and CD103 expression and time after transplant, in intestinal transplant grafts (n = 21; 12 subjects). MFI represented by bars. (C–H) Cytokine production by small intestinal CD4 + T cells. Representative histograms of expression, and group summaries of proportion of CD4 + T cells expressing IL-17A (C), TNF-α (D), IFN-γ (E), IL-2 (F), CCL4 (G), and IL-10 (H) after 4 h stimulation with PMA and ionomycin in the presence of brefeldin A and monensin, categorized by CD69 and CD103 expression, in small intestinal biopsies from healthy control subjects (n = 10). (I) Mean proportion of CD4 + T cells expressing 0, 1, 2, 3, 4, 5, or 6 of the cytokines or chemokines IL-17A, TNF-α, IFN-γ, CCL4, IL-2, and IL-10, categorized by CD69 and CD103 co-expression, from small intestinal biopsies from healthy control subjects (n = 10). Statistical analysis performed with one-way ANOVA with Tukey’s multiple-comparison test. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001.
    Figure Legend Snippet: CD103 + and CD103 − CD4 + T cells display analogous phenotypic and functional differences to their CD8 + counterparts (A) MFI (CD161, CD7, CD127, β2-integrin, granzyme K, and KLRG1) or percentage positive (Ki-67) of CD4 + T cells, categorized by CD69 and CD103 expression, in small intestinal biopsies from healthy control subjects (n = 10). MFI represented by bars. Connecting lines represent populations from the same subject. Blue, CD69 − CD103 − cells; red, CD69 + CD103 − cells; black, CD69 + CD103 + cells. (B) MFI of β2-integrin on recipient-derived CD4 + T cells, categorized by CD69 and CD103 expression and time after transplant, in intestinal transplant grafts (n = 21; 12 subjects). MFI represented by bars. (C–H) Cytokine production by small intestinal CD4 + T cells. Representative histograms of expression, and group summaries of proportion of CD4 + T cells expressing IL-17A (C), TNF-α (D), IFN-γ (E), IL-2 (F), CCL4 (G), and IL-10 (H) after 4 h stimulation with PMA and ionomycin in the presence of brefeldin A and monensin, categorized by CD69 and CD103 expression, in small intestinal biopsies from healthy control subjects (n = 10). (I) Mean proportion of CD4 + T cells expressing 0, 1, 2, 3, 4, 5, or 6 of the cytokines or chemokines IL-17A, TNF-α, IFN-γ, CCL4, IL-2, and IL-10, categorized by CD69 and CD103 co-expression, from small intestinal biopsies from healthy control subjects (n = 10). Statistical analysis performed with one-way ANOVA with Tukey’s multiple-comparison test. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001.

    Techniques Used: Functional Assay, Expressing, Derivative Assay

    CD103 + CD8 + intestinal T cells demonstrate greater capacity for cytokine production (A–D) Cytokine production by small intestinal CD8 + T cells. Representative histograms of expression, and group summaries of proportion of CD8 + T cells expressing TNF-α (A), IFN-γ (B), CCL4 (C), and IL-2 (D) after 4 h stimulation with PMA and ionomycin in the presence of brefeldin A and monensin, categorized by CD69 and CD103 expression, in small intestinal biopsies from healthy control subjects (n = 10). (E) Mean proportion of CD8 + T cells expressing 0, 1, 2, 3, or 4 of the cytokines/chemokines TNF-α, IFN-γ, CCL4, and IL-2, categorized by CD69 and CD103 co-expression, from small intestinal biopsies from healthy control subjects (n = 10). (F) Mean percentage (± SEM) of CD8 + T cells co-expressing TNF-α, IFN-γ, CCL4, and/or IL-2 after PMA and ionomycin stimulation as described, categorized by CD69 and CD103 expression. Statistical analysis performed with one-way ANOVA with Tukey’s multiple-comparison test. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001.
    Figure Legend Snippet: CD103 + CD8 + intestinal T cells demonstrate greater capacity for cytokine production (A–D) Cytokine production by small intestinal CD8 + T cells. Representative histograms of expression, and group summaries of proportion of CD8 + T cells expressing TNF-α (A), IFN-γ (B), CCL4 (C), and IL-2 (D) after 4 h stimulation with PMA and ionomycin in the presence of brefeldin A and monensin, categorized by CD69 and CD103 expression, in small intestinal biopsies from healthy control subjects (n = 10). (E) Mean proportion of CD8 + T cells expressing 0, 1, 2, 3, or 4 of the cytokines/chemokines TNF-α, IFN-γ, CCL4, and IL-2, categorized by CD69 and CD103 co-expression, from small intestinal biopsies from healthy control subjects (n = 10). (F) Mean percentage (± SEM) of CD8 + T cells co-expressing TNF-α, IFN-γ, CCL4, and/or IL-2 after PMA and ionomycin stimulation as described, categorized by CD69 and CD103 expression. Statistical analysis performed with one-way ANOVA with Tukey’s multiple-comparison test. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001.

    Techniques Used: Expressing

    3) Product Images from "STAT5 promotes accessibility and is required for BATF-mediated plasticity at the Il9 locus"

    Article Title: STAT5 promotes accessibility and is required for BATF-mediated plasticity at the Il9 locus

    Journal: Nature Communications

    doi: 10.1038/s41467-020-18648-6

    Cooperation between STAT5 and BATF in the plasticity of the Il9 locus. Naive CD4+ T cells were isolated from the spleen and differentiated into Th lineage for 5 days. Retrovirus expressing caSTAT5 (tagged with hNGFR), BATF (tagged with Thy1.1), or IRF4 (tagged with GFP) was transduced on day 1. For cytokine production analysis, cells were stimulated with PMA/Ionomycin for 5 h and monensin was added for the last 2 h. a Venn diagram of overlap between BATF and STAT5b target genes in Th9 cells. b , c Flow cytometric analysis of cytokine expression in Th17 cells co-transduced with caSTAT5 and BATF retrovirus. d Flow cytometric analysis of IL-9 expression in Th17 cells co-transduced with caSTAT5 and IRF4 retrovirus; cells were analyzed on day 5. e , f Flow cytometric analysis of cytokine expression in WT or Batf ΔZ/ΔZ Th9 cells co-transduced with caSTAT5 and BATF retrovirus; cells were analyzed on day 5. g , h Flow cytometric analysis of cytokine expression in WT or Batf ΔZ/ΔZ Th17 cells co-transduced with caSTAT5 and BATF retrovirus; cells were analyzed on day 5. i Flow cytometric analysis of IL-9 expression in caSTAT5 lone or caSTAT5 and BATF retrovirus co-transduced Th0, Th1, Th2, and Treg cells, cells were analyzed on day 5. Date are mean ± SEM of three mice per experiment and representative of two independent experiments. Two-way ANOVA with Sidak’s multiple comparison test was used to generate p -values. ns: no significant, p > 0.05. See also Supplementary Fig. 4 .
    Figure Legend Snippet: Cooperation between STAT5 and BATF in the plasticity of the Il9 locus. Naive CD4+ T cells were isolated from the spleen and differentiated into Th lineage for 5 days. Retrovirus expressing caSTAT5 (tagged with hNGFR), BATF (tagged with Thy1.1), or IRF4 (tagged with GFP) was transduced on day 1. For cytokine production analysis, cells were stimulated with PMA/Ionomycin for 5 h and monensin was added for the last 2 h. a Venn diagram of overlap between BATF and STAT5b target genes in Th9 cells. b , c Flow cytometric analysis of cytokine expression in Th17 cells co-transduced with caSTAT5 and BATF retrovirus. d Flow cytometric analysis of IL-9 expression in Th17 cells co-transduced with caSTAT5 and IRF4 retrovirus; cells were analyzed on day 5. e , f Flow cytometric analysis of cytokine expression in WT or Batf ΔZ/ΔZ Th9 cells co-transduced with caSTAT5 and BATF retrovirus; cells were analyzed on day 5. g , h Flow cytometric analysis of cytokine expression in WT or Batf ΔZ/ΔZ Th17 cells co-transduced with caSTAT5 and BATF retrovirus; cells were analyzed on day 5. i Flow cytometric analysis of IL-9 expression in caSTAT5 lone or caSTAT5 and BATF retrovirus co-transduced Th0, Th1, Th2, and Treg cells, cells were analyzed on day 5. Date are mean ± SEM of three mice per experiment and representative of two independent experiments. Two-way ANOVA with Sidak’s multiple comparison test was used to generate p -values. ns: no significant, p > 0.05. See also Supplementary Fig. 4 .

    Techniques Used: Isolation, Expressing, Transduction, Mouse Assay

    Accessibility is required for BATF to activate Il9 . Naive CD4+ T cells were isolated from spleen and differentiated into Th9 and Th17 cells for 5 days ( a – c ) or 6 days ( d – h ). dCas9/dCas9-VP64- and gRNA-expressing retrovirus were transduced on day 1; BATF, IRF4, and control vector was transduced on day 4. ChIP assay and chromatin accessibility assay were performed on day 5 or day 6. For cytokine production analysis, Th9 or Th17 cells were stimulated with phorbol 12-myristate 13-acetate (PMA)/Ionomycin for 5 h and monensin was added for the last 2 h. a Flow cytometry analysis of IL-9 production in WT, Batf ΔZ/ΔZ , and Stat6 −/− Th9 cells on day 5 ( n = 3 per group). b Binding of RNA polymerase II on the Il9 promoter as determined by ChIP-qPCR on day 5 ( n = 3 per group). c Chromatin accessibility analysis of Il9 locus in WT, Batf ΔZ/ΔZ , and Stat6 −/− Th9 cells on day 5 ( n = 3 per group). d Th17 cells transduced with control vectors or dCas9-VP64 and gRNA-CNS1 were sorted and chromatin accessibility of Il9 locus was detected on day 6 ( n = 4 for control vector group, n = 3 for dCas9-VP64 gRNA-CNS1 group). e Th17 cells were transduced with dCas9-VP64 and gRNA-CNS1, followed by BATF or control vector transduction. Transduced cells were sorted on day 6 and chromatin accessibility of the Il9 locus was assessed ( n = 4 for dCas9 gRNA-CNS1 group, n = 3 for dCas9-VP64 gRNA-CNS1 group). f Flow cytometry analysis of IL-9 expression in Th17 cells transduced with retrovirus expressing gRNA-CNS1, dCas9-VP64, BATF, or control vectors on day 6 ( n = 3 per group). g Flow cytometry analysis of IL-9 expression in Th17 cells transduced with retrovirus expressing gRNA-CNS-25, dCas9-VP64, BATF, or control vectors on day 6 ( n = 3 per group). h Flow cytometry analysis of IL-9 expression in Th17 cells transduced with retrovirus expressing gRNA-CNS-25, dCas9-VP64, IRF4, or control vectors on day 6 ( n = 3 per group). Histograms are gated on transduced CD4+ T cells. Data are mean ± SEM. a , b One-way ANOVA with a Dunnett’s multiple comparison test was used to generate p -values for multiple comparisons. c Unpaired two-tailed Student’s t -test was used for comparison. f , g Two-way ANOVA with Sidak’s multiple comparisons was used to generate p -values. See also Supplementary Fig. 2 .
    Figure Legend Snippet: Accessibility is required for BATF to activate Il9 . Naive CD4+ T cells were isolated from spleen and differentiated into Th9 and Th17 cells for 5 days ( a – c ) or 6 days ( d – h ). dCas9/dCas9-VP64- and gRNA-expressing retrovirus were transduced on day 1; BATF, IRF4, and control vector was transduced on day 4. ChIP assay and chromatin accessibility assay were performed on day 5 or day 6. For cytokine production analysis, Th9 or Th17 cells were stimulated with phorbol 12-myristate 13-acetate (PMA)/Ionomycin for 5 h and monensin was added for the last 2 h. a Flow cytometry analysis of IL-9 production in WT, Batf ΔZ/ΔZ , and Stat6 −/− Th9 cells on day 5 ( n = 3 per group). b Binding of RNA polymerase II on the Il9 promoter as determined by ChIP-qPCR on day 5 ( n = 3 per group). c Chromatin accessibility analysis of Il9 locus in WT, Batf ΔZ/ΔZ , and Stat6 −/− Th9 cells on day 5 ( n = 3 per group). d Th17 cells transduced with control vectors or dCas9-VP64 and gRNA-CNS1 were sorted and chromatin accessibility of Il9 locus was detected on day 6 ( n = 4 for control vector group, n = 3 for dCas9-VP64 gRNA-CNS1 group). e Th17 cells were transduced with dCas9-VP64 and gRNA-CNS1, followed by BATF or control vector transduction. Transduced cells were sorted on day 6 and chromatin accessibility of the Il9 locus was assessed ( n = 4 for dCas9 gRNA-CNS1 group, n = 3 for dCas9-VP64 gRNA-CNS1 group). f Flow cytometry analysis of IL-9 expression in Th17 cells transduced with retrovirus expressing gRNA-CNS1, dCas9-VP64, BATF, or control vectors on day 6 ( n = 3 per group). g Flow cytometry analysis of IL-9 expression in Th17 cells transduced with retrovirus expressing gRNA-CNS-25, dCas9-VP64, BATF, or control vectors on day 6 ( n = 3 per group). h Flow cytometry analysis of IL-9 expression in Th17 cells transduced with retrovirus expressing gRNA-CNS-25, dCas9-VP64, IRF4, or control vectors on day 6 ( n = 3 per group). Histograms are gated on transduced CD4+ T cells. Data are mean ± SEM. a , b One-way ANOVA with a Dunnett’s multiple comparison test was used to generate p -values for multiple comparisons. c Unpaired two-tailed Student’s t -test was used for comparison. f , g Two-way ANOVA with Sidak’s multiple comparisons was used to generate p -values. See also Supplementary Fig. 2 .

    Techniques Used: Isolation, Expressing, Plasmid Preparation, Chromatin Immunoprecipitation, Flow Cytometry, Binding Assay, Real-time Polymerase Chain Reaction, Transduction, Two Tailed Test

    STAT5 promotes the accessibility of IL9 gene for BATF binding in human Th9 cells. Naive CD4+ cells were isolated from human peripheral blood mononuclear cells; cells were differentiated to Th9 cells ( a – i ). ChIP assay and chromatin accessibility assay were performed on day 5. STAT5 inhibitor was added to the culture on day 1. For cytokine production analysis, cells were stimulated with PMA/Ionomycin for 5 h and monensin was added for the last 2 h. For j – m , peripheral blood mononuclear cells or sorted T cells from pediatric asthmatic or non-asthmatic dermatitis patients were analyzed as described. a , b Kinetic analysis of pSTAT5 detection from naive human CD4+ cells to D5 Th9 culture ( n = 3). c Kinetic analysis of IL-9 expression from D0 to D5 Th9 culture ( n = 4 for D0 to D2 groups, n = 3 for D3 to D5 groups). d , e Kinetic analysis of IL9 gene accessibility from naive human CD4+ cells to D5 Th9 culture ( n = 3). f IL-9 expression in Th9 cells transduced with Scr-shRNA or STAT5b-shRNA lentivirus, cells were analyzed on day 5 ( n = 4). g Th9 cells treated with DMSO or STAT5 inhibitor on day 1, IL-9 and pSTAT5 were analyzed on day 5 ( n = 4). h Chromatin accessibility analysis of IL9 gene locus in Th9 cells treated with DMSO or STAT5 inhibitor; cells were analyzed on day 5 ( n = 3). i The binding of BATF at the IL9 gene locus in Th9 cells treated with DMSO or STAT5 inhibitor ( n = 3). j ELISA analysis of IL-9 expression in the peripheral blood mononuclear cells from patient samples stimulated with anti-CD3 for 12 h ( n = 4 for non-asthma group, n = 6 for asthma group). k Flow cytometric analysis of CCR4+CD4+ cells on peripheral blood mononuclear cells from non-asthma or asthma patients ( n = 4 for non-asthma group, n = 6 for asthma group). l Flow cytometric analysis of pSTAT5 expression cells in peripheral blood mononuclear cells from non-asthma or asthma patients stimulated with 100 U/ml IL-2 for 10 min ( n = 4 for non-asthma group, n = 7 for asthma group). m Chromatin accessibility analysis of the IL9 gene locus in CCR4+CD4+ cells sorted from patient peripheral blood mononuclear cells ( n = 4 for non-asthma group, n = 3 for asthma group). Date are mean ± SEM. One-way ANOVA with a Dunnett’s multiple comparison test was used to generate p -values for all multiple comparisons in b – e . An unpaired two-tailed Student t-test was used for comparisons in f – m .
    Figure Legend Snippet: STAT5 promotes the accessibility of IL9 gene for BATF binding in human Th9 cells. Naive CD4+ cells were isolated from human peripheral blood mononuclear cells; cells were differentiated to Th9 cells ( a – i ). ChIP assay and chromatin accessibility assay were performed on day 5. STAT5 inhibitor was added to the culture on day 1. For cytokine production analysis, cells were stimulated with PMA/Ionomycin for 5 h and monensin was added for the last 2 h. For j – m , peripheral blood mononuclear cells or sorted T cells from pediatric asthmatic or non-asthmatic dermatitis patients were analyzed as described. a , b Kinetic analysis of pSTAT5 detection from naive human CD4+ cells to D5 Th9 culture ( n = 3). c Kinetic analysis of IL-9 expression from D0 to D5 Th9 culture ( n = 4 for D0 to D2 groups, n = 3 for D3 to D5 groups). d , e Kinetic analysis of IL9 gene accessibility from naive human CD4+ cells to D5 Th9 culture ( n = 3). f IL-9 expression in Th9 cells transduced with Scr-shRNA or STAT5b-shRNA lentivirus, cells were analyzed on day 5 ( n = 4). g Th9 cells treated with DMSO or STAT5 inhibitor on day 1, IL-9 and pSTAT5 were analyzed on day 5 ( n = 4). h Chromatin accessibility analysis of IL9 gene locus in Th9 cells treated with DMSO or STAT5 inhibitor; cells were analyzed on day 5 ( n = 3). i The binding of BATF at the IL9 gene locus in Th9 cells treated with DMSO or STAT5 inhibitor ( n = 3). j ELISA analysis of IL-9 expression in the peripheral blood mononuclear cells from patient samples stimulated with anti-CD3 for 12 h ( n = 4 for non-asthma group, n = 6 for asthma group). k Flow cytometric analysis of CCR4+CD4+ cells on peripheral blood mononuclear cells from non-asthma or asthma patients ( n = 4 for non-asthma group, n = 6 for asthma group). l Flow cytometric analysis of pSTAT5 expression cells in peripheral blood mononuclear cells from non-asthma or asthma patients stimulated with 100 U/ml IL-2 for 10 min ( n = 4 for non-asthma group, n = 7 for asthma group). m Chromatin accessibility analysis of the IL9 gene locus in CCR4+CD4+ cells sorted from patient peripheral blood mononuclear cells ( n = 4 for non-asthma group, n = 3 for asthma group). Date are mean ± SEM. One-way ANOVA with a Dunnett’s multiple comparison test was used to generate p -values for all multiple comparisons in b – e . An unpaired two-tailed Student t-test was used for comparisons in f – m .

    Techniques Used: Binding Assay, Isolation, Chromatin Immunoprecipitation, Expressing, Transduction, shRNA, Enzyme-linked Immunosorbent Assay, Two Tailed Test

    STAT5 regulates Il9 chromatin accessibility. Naive CD4+ T cells were isolated from the spleen and differentiated into Th9 and Th17 cells for 5 days. ChIP assay and chromatin accessibility assay were performed on day 5. shRNA-expressing retrovirus was transduced on day 1. For cytokine production analysis, cells were stimulated with PMA/Ionomycin for 5 h and monensin was added for the last 2 h. a – c Kinetic analysis of chromatin accessibility, transcription factors, and chromatin modification markers binding on Il9 promoter locus in Th9 cells during Th9 differentiation. d FACS analysis of IL-9 and pSTAT5 expression in Th9 cells transduced with control (Scr), STAT5a-specific, or STAT5b-specific shRNA; cells were gated on transduced CD4+ live cells. e Chromatin accessibility analysis of Il9 gene locus in Th9 cells transduced with Scr-shRNA or STAT5a-shRNA retrovirus. f , g H3K27me3 modification and BATF binding at the Il9 gene locus in Th9 cells transduced with Scr-shRNA or STAT5a-shRNA retrovirus on day 5. h , i FACS analysis of IL-9 and IL-17 expression on day 5 in Th17 cells transduced with control vector or caSTAT5 retrovirus; cells were gated on transduced CD4+ live cells. j Chromatin accessibility analysis of Il9 gene locus in Th17 cells that transduced with control vector or caSTAT5 retrovirus; transduced cells were sorted on day 5. k The binding of BATF on Il9 gene locus in Th17 cells that transduced with control vector or caSTAT5 retrovirus; transduced cells were sorted on day 5. Date are mean ± SEM of three mice per experiment and representative of two independent experiments. The p -values for a and b are compared to D0 for chromatin accessibility and STAT5 binding; D1 for other transcription factors and chromatin modification markers binding. One-way ANOVA with a post hoc Tukey’s test was used to generate p -values for all multiple comparisons in b . An unpaired two-tailed Student’s t -test was used for comparisons in a , d , e , h , j , and k . See also Supplementary Fig. 3 .
    Figure Legend Snippet: STAT5 regulates Il9 chromatin accessibility. Naive CD4+ T cells were isolated from the spleen and differentiated into Th9 and Th17 cells for 5 days. ChIP assay and chromatin accessibility assay were performed on day 5. shRNA-expressing retrovirus was transduced on day 1. For cytokine production analysis, cells were stimulated with PMA/Ionomycin for 5 h and monensin was added for the last 2 h. a – c Kinetic analysis of chromatin accessibility, transcription factors, and chromatin modification markers binding on Il9 promoter locus in Th9 cells during Th9 differentiation. d FACS analysis of IL-9 and pSTAT5 expression in Th9 cells transduced with control (Scr), STAT5a-specific, or STAT5b-specific shRNA; cells were gated on transduced CD4+ live cells. e Chromatin accessibility analysis of Il9 gene locus in Th9 cells transduced with Scr-shRNA or STAT5a-shRNA retrovirus. f , g H3K27me3 modification and BATF binding at the Il9 gene locus in Th9 cells transduced with Scr-shRNA or STAT5a-shRNA retrovirus on day 5. h , i FACS analysis of IL-9 and IL-17 expression on day 5 in Th17 cells transduced with control vector or caSTAT5 retrovirus; cells were gated on transduced CD4+ live cells. j Chromatin accessibility analysis of Il9 gene locus in Th17 cells that transduced with control vector or caSTAT5 retrovirus; transduced cells were sorted on day 5. k The binding of BATF on Il9 gene locus in Th17 cells that transduced with control vector or caSTAT5 retrovirus; transduced cells were sorted on day 5. Date are mean ± SEM of three mice per experiment and representative of two independent experiments. The p -values for a and b are compared to D0 for chromatin accessibility and STAT5 binding; D1 for other transcription factors and chromatin modification markers binding. One-way ANOVA with a post hoc Tukey’s test was used to generate p -values for all multiple comparisons in b . An unpaired two-tailed Student’s t -test was used for comparisons in a , d , e , h , j , and k . See also Supplementary Fig. 3 .

    Techniques Used: Isolation, Chromatin Immunoprecipitation, shRNA, Expressing, Modification, Binding Assay, FACS, Transduction, Plasmid Preparation, Mouse Assay, Two Tailed Test

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    Article Title: In vivo depletion of CD4+CD25hi regulatory T cells is associated with improved antiviral responses in cats chronically infected with feline immunodeficiency virus
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    Article Title: Partial Regulatory T Cell Depletion Prior to Acute Feline Immunodeficiency Virus Infection Does Not Alter Disease Pathogenesis
    Article Snippet: Anti-mouse IgG3-APC (Jackson ImmunoResearch, West Grove, PA), anti-mouse IgG3-APC-Cy7 (Southern Biotech), and streptavidin-Pacific Orange (Invitrogen) were used for secondary detection. .. Intracellular FoxP3 staining was performed with eBioscience FoxP3 staining buffers and FoxP3-PE-Cy7 (FJK-16s; San Diego, CA) according to manufacturer's protocol, with the exception that cells remain in the permeabilization/wash buffer no longer than 30 min. For intracellular cytokine staining, cells were incubated with 1× monensin (BioLegend, San Diego, CA) for six hr, labeled for surface markers, fixed with 4% paraformaldehyde, permeabilized with BD Cytofix/Cytoperm kit Perm/Wash buffer, and stained with anti-cytokine mAbs. .. For intracellular Ki67 staining, cells were first labeled for surface markers, incubated with BD Cytofix/Cytoperm for 15 min, washed with BD Perm/Wash buffer, incubated with BD Cytoperm Plus for 10 min, washed, and incubated with BD Cytofix/Cytoperm for 5 min.

    Staining:

    Article Title: In vivo depletion of CD4+CD25hi regulatory T cells is associated with improved antiviral responses in cats chronically infected with feline immunodeficiency virus
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    Article Title: Inhibition of Ubc13-mediated Ubiquitination by GPS2 Regulates Multiple Stages of B Cell Development *
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    Article Title: STAT5 promotes accessibility and is required for BATF-mediated plasticity at the Il9 locus
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    Labeling:

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    Recombinant:

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    BioLegend protein transport inhibitor monensin
    Blocking IL-10 signalling at the time of long HPV16 E7 peptide/MPLA immunization increases the numbers of IL-10 producing cells. Groups of five C57BL/6 mice were immunized as indicated with 50 μg of E7 peptide/15 μg of Monophosphoryl Lipid A (MPLA), 300 μg of anti-IL10R antibodies or control antibodies s.c on days 0 and 14. Splenocytes and draining lymph node cells from immunized mice were harvested and stimulated with 25 ng/ml PMA and 1 μg/ml ionomycin for 6 h in the presence of <t>monensin</t> on 7 days after final immunization. Cells were surface stained for CD3, CD4, and GITR and intracellular stained for IL-10 and IFN-γ. CD3+ cells were gated. Results for cells of draining lymph nodes were shown. A and B: IL-10 secreting CD3 + CD4+ T cells IL-10 ( a ) FACS plot and ( b ) summarised data showing IL-10 expression by CD3 + CD4+ T cells. C and D: CD3 + CD4+ T cells secreting IL-10 and IFN-γ dot plots ( c ) and summarised data from different groups ( d ) E and F: CD4 + GITR+ T cells secreting IL-10: FACS profile ( e ) and summarised data from different groups ( f ). Splenic CD4 + IL-10+ cells were shown in ( g )
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    Blocking IL-10 signalling at the time of long HPV16 E7 peptide/MPLA immunization increases the numbers of IL-10 producing cells. Groups of five C57BL/6 mice were immunized as indicated with 50 μg of E7 peptide/15 μg of Monophosphoryl Lipid A (MPLA), 300 μg of anti-IL10R antibodies or control antibodies s.c on days 0 and 14. Splenocytes and draining lymph node cells from immunized mice were harvested and stimulated with 25 ng/ml PMA and 1 μg/ml ionomycin for 6 h in the presence of monensin on 7 days after final immunization. Cells were surface stained for CD3, CD4, and GITR and intracellular stained for IL-10 and IFN-γ. CD3+ cells were gated. Results for cells of draining lymph nodes were shown. A and B: IL-10 secreting CD3 + CD4+ T cells IL-10 ( a ) FACS plot and ( b ) summarised data showing IL-10 expression by CD3 + CD4+ T cells. C and D: CD3 + CD4+ T cells secreting IL-10 and IFN-γ dot plots ( c ) and summarised data from different groups ( d ) E and F: CD4 + GITR+ T cells secreting IL-10: FACS profile ( e ) and summarised data from different groups ( f ). Splenic CD4 + IL-10+ cells were shown in ( g )

    Journal: BMC Immunology

    Article Title: Blocking IL-10 signalling at the time of immunization does not increase unwanted side effects in mice

    doi: 10.1186/s12865-017-0224-x

    Figure Lengend Snippet: Blocking IL-10 signalling at the time of long HPV16 E7 peptide/MPLA immunization increases the numbers of IL-10 producing cells. Groups of five C57BL/6 mice were immunized as indicated with 50 μg of E7 peptide/15 μg of Monophosphoryl Lipid A (MPLA), 300 μg of anti-IL10R antibodies or control antibodies s.c on days 0 and 14. Splenocytes and draining lymph node cells from immunized mice were harvested and stimulated with 25 ng/ml PMA and 1 μg/ml ionomycin for 6 h in the presence of monensin on 7 days after final immunization. Cells were surface stained for CD3, CD4, and GITR and intracellular stained for IL-10 and IFN-γ. CD3+ cells were gated. Results for cells of draining lymph nodes were shown. A and B: IL-10 secreting CD3 + CD4+ T cells IL-10 ( a ) FACS plot and ( b ) summarised data showing IL-10 expression by CD3 + CD4+ T cells. C and D: CD3 + CD4+ T cells secreting IL-10 and IFN-γ dot plots ( c ) and summarised data from different groups ( d ) E and F: CD4 + GITR+ T cells secreting IL-10: FACS profile ( e ) and summarised data from different groups ( f ). Splenic CD4 + IL-10+ cells were shown in ( g )

    Article Snippet: Intracellular staining for IFNγ, IL-10 Single spleen cell suspensions or single lymph node cell suspension obtained from immunized and control mice were stimulated with PMA and ionomycin for 3–5 h in the presence of protein transport inhibitor monensin (BioLegend).

    Techniques: Blocking Assay, Mouse Assay, Staining, FACS, Expressing

    c-Met is stably palmitoylated in the ER H1993 cells were incubated with azido-homoalanine (AHA) A. or Odya B. alone or with cycloheximide (CX, 10 μg/ml), brefeldin A (BF, 2 μM), or monensin (MN, 2 μM) for 30, 60, or 120 mins (m). C. DU145 cells were incubated with Odya for 2 hours (T0) before chase periods of 20 or 40 minutes in the presence of Odya +/− HGF. For each, the click reaction was performed followed by biotin pull-down and western blot analysis to detect the presence of c-Met. Representative blots from three independent experiments are shown.

    Journal: Oncotarget

    Article Title: Palmitoylation regulates the intracellular trafficking and stability of c-Met

    doi: 10.18632/oncotarget.8706

    Figure Lengend Snippet: c-Met is stably palmitoylated in the ER H1993 cells were incubated with azido-homoalanine (AHA) A. or Odya B. alone or with cycloheximide (CX, 10 μg/ml), brefeldin A (BF, 2 μM), or monensin (MN, 2 μM) for 30, 60, or 120 mins (m). C. DU145 cells were incubated with Odya for 2 hours (T0) before chase periods of 20 or 40 minutes in the presence of Odya +/− HGF. For each, the click reaction was performed followed by biotin pull-down and western blot analysis to detect the presence of c-Met. Representative blots from three independent experiments are shown.

    Article Snippet: Monensin and brefeldin A were obtained from Biolegend (San Diego, CA), Y27637 from Stem Cell Technology (Vancouver, BC), C75, 2-hydroxymyristic acid, and 17-Octadecynoic Acid from Caymen Chemicals (Ann Arbor, MI), cycloheximide, recombinant EGF, geranylgeranyltransferase inhibitor, farnesyltransferase inhibitor, vinblastine, latrunculin, dansylcadaverine, ethylisopropyl amiloride, methyl-β-cyclodextrin, nystatin, and 2-bromopalmitate were obtained from Sigma-Aldrich (St. Louis, MO).

    Techniques: Stable Transfection, Incubation, Western Blot

    Anti-CD25 treatment does not result in elevated constitutive T cell IL-2 or TNF-α production Intracellular IL-2 and TNF-α staining was performed on lymphocytes after a 6 hour incubation with 1x monensin, surface phenotype staining, and fixation with 2% paraformaldehyde. The cells were permeabilized with BD Perm/Wash buffer. (A) IL-2 + and (B) TNF-α + cells per million CD4 + or CD8 + T cells were quantified by flow cytometry in popliteal lymph nodes before mAb treatment and prescapular lymph nodes 23 days after mAb treatment. ■ indicates isotype control group; ▼, CD25 depleted group.

    Journal: Virology

    Article Title: In vivo depletion of CD4+CD25hi regulatory T cells is associated with improved antiviral responses in cats chronically infected with feline immunodeficiency virus

    doi: 10.1016/j.virol.2010.04.016

    Figure Lengend Snippet: Anti-CD25 treatment does not result in elevated constitutive T cell IL-2 or TNF-α production Intracellular IL-2 and TNF-α staining was performed on lymphocytes after a 6 hour incubation with 1x monensin, surface phenotype staining, and fixation with 2% paraformaldehyde. The cells were permeabilized with BD Perm/Wash buffer. (A) IL-2 + and (B) TNF-α + cells per million CD4 + or CD8 + T cells were quantified by flow cytometry in popliteal lymph nodes before mAb treatment and prescapular lymph nodes 23 days after mAb treatment. ■ indicates isotype control group; ▼, CD25 depleted group.

    Article Snippet: Intracellular FOXP3 staining was performed with eBioscience FOXP3 staining buffers and FOXP3-PECy7 (FJK-16s; San Diego, CA) according to manufacturer’s protocol, with the exception that cells remain in the permeabilization/wash buffer no longer than 30 min. For intracellular cytokine staining, cells were incubated with 1x monensin (Biolegend, San Diego, CA) for six hours, labeled for surface markers, fixed with 4% paraformaldehyde, permeabilized with BD Cytofix/Cytoperm kit Perm/Wash buffer, and stained with IL-2-PE (MQ1-17H12; BioLegend) and tumor necrosis factor (TNF)-α-APC (6401.1111; BD).

    Techniques: Staining, Incubation, Flow Cytometry, Cytometry

    Cytokine-expressing CD8 + T cell levels are lower after anti-CD25 mAb treatment on day 35 post-FIV infection. Popliteal lymph node cells were incubated with monensin for 6 hours prior to quantification of cytokine expressing cells. Percent TNF-α-, IL-2-, and IFN-γ-expressing cells in CD4 + and CD8 + T cell populations were quantified by flow cytometry (n = 8/group). Mean ± SEM is shown. * indicates p

    Journal: PLoS ONE

    Article Title: Partial Regulatory T Cell Depletion Prior to Acute Feline Immunodeficiency Virus Infection Does Not Alter Disease Pathogenesis

    doi: 10.1371/journal.pone.0017183

    Figure Lengend Snippet: Cytokine-expressing CD8 + T cell levels are lower after anti-CD25 mAb treatment on day 35 post-FIV infection. Popliteal lymph node cells were incubated with monensin for 6 hours prior to quantification of cytokine expressing cells. Percent TNF-α-, IL-2-, and IFN-γ-expressing cells in CD4 + and CD8 + T cell populations were quantified by flow cytometry (n = 8/group). Mean ± SEM is shown. * indicates p

    Article Snippet: Intracellular FoxP3 staining was performed with eBioscience FoxP3 staining buffers and FoxP3-PE-Cy7 (FJK-16s; San Diego, CA) according to manufacturer's protocol, with the exception that cells remain in the permeabilization/wash buffer no longer than 30 min. For intracellular cytokine staining, cells were incubated with 1× monensin (BioLegend, San Diego, CA) for six hr, labeled for surface markers, fixed with 4% paraformaldehyde, permeabilized with BD Cytofix/Cytoperm kit Perm/Wash buffer, and stained with anti-cytokine mAbs.

    Techniques: Expressing, Infection, Incubation, Flow Cytometry, Cytometry