brefeldin a  (Thermo Fisher)


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    Structured Review

    Thermo Fisher brefeldin a
    TGN clathrin adaptor localization is unaffected by deletion of LDB19 . (A) Wild-type and ldb19Δ cells expressing Gga2-GFP or Apl2-GFP were imaged. (B) Quantification of the fluorescence intensities of the punctate structures from the images in A. Graphs are of data points measured from a representative experiment showing the median and interquartile ranges (25 and 75 percentile). Each data point corresponds to a single punctate structure. AU, arbitrary units. (C) Western blot showing the protein levels of clathrin adaptor proteins in WT and ldb19Δ cells. Adh1 was used as a lysis control. Data is representative of 3 repeats. (D), (E) Wild-type and gga1Δgga2Δ or apl2Δ cells expressing Ldb19-GFP were imaged. Right, quantification of Ldb19-GFP punctate structures as in B. Non parametric Mann Whitney test was performed (****p > 0.0001). (F) erg6 Δ cells expressing Ldb19-GFP and Gga2-mCh were imaged following treatment with DMSO (no BFA) or 150μM <t>Brefeldin</t> A (+BFA). Arrowhead points to rare puncta in treated cells. (G) Quantification of the percentage cells from F that contain Ldb19-GFP puncta in the absence or presence of BFA. Non parametric t-test was performed (****p
    Brefeldin A, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1338 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Investigation of Ldb19/Art1 localization and function at the late Golgi"

    Article Title: Investigation of Ldb19/Art1 localization and function at the late Golgi

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0206944

    TGN clathrin adaptor localization is unaffected by deletion of LDB19 . (A) Wild-type and ldb19Δ cells expressing Gga2-GFP or Apl2-GFP were imaged. (B) Quantification of the fluorescence intensities of the punctate structures from the images in A. Graphs are of data points measured from a representative experiment showing the median and interquartile ranges (25 and 75 percentile). Each data point corresponds to a single punctate structure. AU, arbitrary units. (C) Western blot showing the protein levels of clathrin adaptor proteins in WT and ldb19Δ cells. Adh1 was used as a lysis control. Data is representative of 3 repeats. (D), (E) Wild-type and gga1Δgga2Δ or apl2Δ cells expressing Ldb19-GFP were imaged. Right, quantification of Ldb19-GFP punctate structures as in B. Non parametric Mann Whitney test was performed (****p > 0.0001). (F) erg6 Δ cells expressing Ldb19-GFP and Gga2-mCh were imaged following treatment with DMSO (no BFA) or 150μM Brefeldin A (+BFA). Arrowhead points to rare puncta in treated cells. (G) Quantification of the percentage cells from F that contain Ldb19-GFP puncta in the absence or presence of BFA. Non parametric t-test was performed (****p
    Figure Legend Snippet: TGN clathrin adaptor localization is unaffected by deletion of LDB19 . (A) Wild-type and ldb19Δ cells expressing Gga2-GFP or Apl2-GFP were imaged. (B) Quantification of the fluorescence intensities of the punctate structures from the images in A. Graphs are of data points measured from a representative experiment showing the median and interquartile ranges (25 and 75 percentile). Each data point corresponds to a single punctate structure. AU, arbitrary units. (C) Western blot showing the protein levels of clathrin adaptor proteins in WT and ldb19Δ cells. Adh1 was used as a lysis control. Data is representative of 3 repeats. (D), (E) Wild-type and gga1Δgga2Δ or apl2Δ cells expressing Ldb19-GFP were imaged. Right, quantification of Ldb19-GFP punctate structures as in B. Non parametric Mann Whitney test was performed (****p > 0.0001). (F) erg6 Δ cells expressing Ldb19-GFP and Gga2-mCh were imaged following treatment with DMSO (no BFA) or 150μM Brefeldin A (+BFA). Arrowhead points to rare puncta in treated cells. (G) Quantification of the percentage cells from F that contain Ldb19-GFP puncta in the absence or presence of BFA. Non parametric t-test was performed (****p

    Techniques Used: Expressing, Fluorescence, Western Blot, Lysis, MANN-WHITNEY

    2) Product Images from "Inactivation of TRPM7 kinase in mice results in enlarged spleens, reduced T-cell proliferation and diminished store-operated calcium entry"

    Article Title: Inactivation of TRPM7 kinase in mice results in enlarged spleens, reduced T-cell proliferation and diminished store-operated calcium entry

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-21004-w

    Expression of IL-2 and TNF-α cytokines in activated KD CD4 + T cells is reduced. ( A , B ) Expression of IL-2 and TNF-α in WT and KD CD4 + splenic T cells, left unstimulated or stimulated for 6 hours with PMA/ionomycin, in the presence of Brefeldin A during the last 5 hrs, determined by flow cytometry. ( C ) Representative flow plots from one independent experiment for data shown in ( B ). ( D , E ) IL-2 and TNF-α expression in resting cells and 16 hrs after PMA/ionomycin stimulation of CD4 + T cells. Brefeldin A was present during the last 6 hrs. For ( A - E ), data were collected from three independent experiments performed on samples from 3 WT and 3 KD mice. Horizontal lines represent arithmetic means. The p value for the differences in the percentage of IL-2 + and TNF-α + cells in WT and KD CD4 + T cells in activated conditions were p = 0.007 ( A ), p = 0.225 ( B ), 0.058 ( D ) and 0.061 ( E ), as determined by Student’s t-test. For resting T cells, the differences between WT and KD in A, B, D and E were not statistically significant.
    Figure Legend Snippet: Expression of IL-2 and TNF-α cytokines in activated KD CD4 + T cells is reduced. ( A , B ) Expression of IL-2 and TNF-α in WT and KD CD4 + splenic T cells, left unstimulated or stimulated for 6 hours with PMA/ionomycin, in the presence of Brefeldin A during the last 5 hrs, determined by flow cytometry. ( C ) Representative flow plots from one independent experiment for data shown in ( B ). ( D , E ) IL-2 and TNF-α expression in resting cells and 16 hrs after PMA/ionomycin stimulation of CD4 + T cells. Brefeldin A was present during the last 6 hrs. For ( A - E ), data were collected from three independent experiments performed on samples from 3 WT and 3 KD mice. Horizontal lines represent arithmetic means. The p value for the differences in the percentage of IL-2 + and TNF-α + cells in WT and KD CD4 + T cells in activated conditions were p = 0.007 ( A ), p = 0.225 ( B ), 0.058 ( D ) and 0.061 ( E ), as determined by Student’s t-test. For resting T cells, the differences between WT and KD in A, B, D and E were not statistically significant.

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

    3) Product Images from "Inactivation of TRPM7 kinase in mice results in enlarged spleens, reduced T-cell proliferation and diminished store-operated calcium entry"

    Article Title: Inactivation of TRPM7 kinase in mice results in enlarged spleens, reduced T-cell proliferation and diminished store-operated calcium entry

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-21004-w

    Expression of IL-2 and TNF-α cytokines in activated KD CD4 + T cells is reduced. ( A , B ) Expression of IL-2 and TNF-α in WT and KD CD4 + splenic T cells, left unstimulated or stimulated for 6 hours with PMA/ionomycin, in the presence of Brefeldin A during the last 5 hrs, determined by flow cytometry. ( C ) Representative flow plots from one independent experiment for data shown in ( B ). ( D , E ) IL-2 and TNF-α expression in resting cells and 16 hrs after PMA/ionomycin stimulation of CD4 + T cells. Brefeldin A was present during the last 6 hrs. For ( A - E ), data were collected from three independent experiments performed on samples from 3 WT and 3 KD mice. Horizontal lines represent arithmetic means. The p value for the differences in the percentage of IL-2 + and TNF-α + cells in WT and KD CD4 + T cells in activated conditions were p = 0.007 ( A ), p = 0.225 ( B ), 0.058 ( D ) and 0.061 ( E ), as determined by Student’s t-test. For resting T cells, the differences between WT and KD in A, B, D and E were not statistically significant.
    Figure Legend Snippet: Expression of IL-2 and TNF-α cytokines in activated KD CD4 + T cells is reduced. ( A , B ) Expression of IL-2 and TNF-α in WT and KD CD4 + splenic T cells, left unstimulated or stimulated for 6 hours with PMA/ionomycin, in the presence of Brefeldin A during the last 5 hrs, determined by flow cytometry. ( C ) Representative flow plots from one independent experiment for data shown in ( B ). ( D , E ) IL-2 and TNF-α expression in resting cells and 16 hrs after PMA/ionomycin stimulation of CD4 + T cells. Brefeldin A was present during the last 6 hrs. For ( A - E ), data were collected from three independent experiments performed on samples from 3 WT and 3 KD mice. Horizontal lines represent arithmetic means. The p value for the differences in the percentage of IL-2 + and TNF-α + cells in WT and KD CD4 + T cells in activated conditions were p = 0.007 ( A ), p = 0.225 ( B ), 0.058 ( D ) and 0.061 ( E ), as determined by Student’s t-test. For resting T cells, the differences between WT and KD in A, B, D and E were not statistically significant.

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

    4) Product Images from "TCDD, FICZ, and Other High Affinity AhR Ligands Dose-Dependently Determine the Fate of CD4+ T Cell Differentiation"

    Article Title: TCDD, FICZ, and Other High Affinity AhR Ligands Dose-Dependently Determine the Fate of CD4+ T Cell Differentiation

    Journal: Toxicological Sciences

    doi: 10.1093/toxsci/kfx215

    Treatment with low, but not high, doses of AhR ligands increases IL-17 production on day 10 of the alloresponse.  (A and B) Spleen and peripheral lymph node cells from B6 mice were transferred into F1 host mice (day 0). Host mice were treated with TCDD (15 μg/kg on day 0), 11-Cl-BBQ (7.5 mg/kg on days 0 and 1, BBQ), ITE (40 mg/kg every 6 h on days 0 and 1), FICZ (10 mg/kg on days 0 and 1), or FICZ (50 μg/kg on days 0 and 1, FICZ low ). On day 2 (A) and day 10 (B), splenocytes were cultured  ex vivo  in the presence of PMA/ionomycin, and IL-17 was measured in the culture supernatant. (C–G) Host mice were treated with low doses of TCDD (0.6 μg/kg on day 0), 11-Cl-BBQ (50 μg/kg on days 0–3, BBQ), ITE (50 μg/kg on days 0–3), or FICZ (50 μg/kg on days 0–3). (C) Mice were weighed from days 6 to 10 to calculate the weight change because of the alloresponse. (D) On day 10, splenocytes were analyzed by flow cytometry for the percentage of donor cells that expressed the CTL phenotype (CD8 + CD44 high CD45RB low ). (E–G) On day 10, splenoyctes were cultured with PMA/ionomycin/brefeldin A/monensin then stained for IL-17. (E) Representative FACS plots gated on donor CD4 +  cells. Percentage of donor cells that were IL-17 low  (F) and IL-17 high  (G). (H) IL-17 was measured in culture supernatants of splenocytes stimulated with PMA/ionomycin. Each point represents an individual mouse. In panel B, groups receiving optimized doses of AhR ligands were analyzed separately from the FICZ low  group due to lack of homoegeneity of variance. Treatment groups with different letters are significantly different.  p  ≤ .05.
    Figure Legend Snippet: Treatment with low, but not high, doses of AhR ligands increases IL-17 production on day 10 of the alloresponse. (A and B) Spleen and peripheral lymph node cells from B6 mice were transferred into F1 host mice (day 0). Host mice were treated with TCDD (15 μg/kg on day 0), 11-Cl-BBQ (7.5 mg/kg on days 0 and 1, BBQ), ITE (40 mg/kg every 6 h on days 0 and 1), FICZ (10 mg/kg on days 0 and 1), or FICZ (50 μg/kg on days 0 and 1, FICZ low ). On day 2 (A) and day 10 (B), splenocytes were cultured ex vivo in the presence of PMA/ionomycin, and IL-17 was measured in the culture supernatant. (C–G) Host mice were treated with low doses of TCDD (0.6 μg/kg on day 0), 11-Cl-BBQ (50 μg/kg on days 0–3, BBQ), ITE (50 μg/kg on days 0–3), or FICZ (50 μg/kg on days 0–3). (C) Mice were weighed from days 6 to 10 to calculate the weight change because of the alloresponse. (D) On day 10, splenocytes were analyzed by flow cytometry for the percentage of donor cells that expressed the CTL phenotype (CD8 + CD44 high CD45RB low ). (E–G) On day 10, splenoyctes were cultured with PMA/ionomycin/brefeldin A/monensin then stained for IL-17. (E) Representative FACS plots gated on donor CD4 + cells. Percentage of donor cells that were IL-17 low (F) and IL-17 high (G). (H) IL-17 was measured in culture supernatants of splenocytes stimulated with PMA/ionomycin. Each point represents an individual mouse. In panel B, groups receiving optimized doses of AhR ligands were analyzed separately from the FICZ low group due to lack of homoegeneity of variance. Treatment groups with different letters are significantly different. p  ≤ .05.

    Techniques Used: Mouse Assay, Cell Culture, Ex Vivo, Flow Cytometry, Cytometry, CTL Assay, Staining, FACS

    5) Product Images from "Toxoplasma Co-infection Prevents Th2 Differentiation and Leads to a Helminth-Specific Th1 Response"

    Article Title: Toxoplasma Co-infection Prevents Th2 Differentiation and Leads to a Helminth-Specific Th1 Response

    Journal: Frontiers in Cellular and Infection Microbiology

    doi: 10.3389/fcimb.2017.00341

    Restricted Th2 responses in co-infected mice. Cells from spleen, mesenteric lymph nodes (mLN), small intestinal lamina propria (siLP) and small intestinal epithelium (siE) were isolated and stimulated with PMA and ionomycin in the presence of Brefeldin A followed by intranuclear staining for the lineage transcription factors GATA3 and T-bet. Gating strategy shown in (A) , Bar graphs showing frequencies of CD4 + T cells expressing GATA3 (B) , T-bet (C) , and T-bet expression in CD8 + T cells (D) . GATA3 expression in the duodeunum of the small intestine with scale bar of 100 μm (E) , data shown as mean ± SEM pooled from 2 independent experiments n = 6, statistical analysis was performed using the Mann-Whitney test. (B–D) shown as mean ± SEM, pooled from two independent experiments with n = 8–10. Statistical analysis was performed using the Kruskal-Wallis with Dunn's multiple comparison test, * P ≤ 0.05, ** P ≤ 0.01, and *** P ≤ 0.001.
    Figure Legend Snippet: Restricted Th2 responses in co-infected mice. Cells from spleen, mesenteric lymph nodes (mLN), small intestinal lamina propria (siLP) and small intestinal epithelium (siE) were isolated and stimulated with PMA and ionomycin in the presence of Brefeldin A followed by intranuclear staining for the lineage transcription factors GATA3 and T-bet. Gating strategy shown in (A) , Bar graphs showing frequencies of CD4 + T cells expressing GATA3 (B) , T-bet (C) , and T-bet expression in CD8 + T cells (D) . GATA3 expression in the duodeunum of the small intestine with scale bar of 100 μm (E) , data shown as mean ± SEM pooled from 2 independent experiments n = 6, statistical analysis was performed using the Mann-Whitney test. (B–D) shown as mean ± SEM, pooled from two independent experiments with n = 8–10. Statistical analysis was performed using the Kruskal-Wallis with Dunn's multiple comparison test, * P ≤ 0.05, ** P ≤ 0.01, and *** P ≤ 0.001.

    Techniques Used: Infection, Mouse Assay, Isolation, Staining, Expressing, MANN-WHITNEY

    Th2 but not Th1 immune responses are absent in co-infected mice. Cells from spleen, mLN, siLP, and siE of single and co-infected animals were isolated and stimulated with PMA and ionomycin in the presence of Brefeldin A followed by intracellular cytokine staining. Gating strategy for the cytokines IL-5 and IFN-γ in CD4 + cells isolated from spleen (A) . Bar graphs showing frequencies of CD4 + T cells expressing IL-4 (B) , IL-5 (C) , IL-13 (D) , and IFN-γ (E) in spleen, mLN, siLP, and siE. (F) IL-4 and IFN-γ production detected by ELISA in supernatant from 3 × 10 5 splenocytes stimulated with (black bars) and without (white bars) anti-CD3/CD28 antibodies. (B–F) Data shown as mean ± SEM, pooled from two independent experiments with n = 8–9 Statistical analysis was performed using the Kruskal-Wallis with Dunn's multiple comparison test, * P ≤ 0.05, ** P ≤ 0.01, and *** P ≤ 0.001.
    Figure Legend Snippet: Th2 but not Th1 immune responses are absent in co-infected mice. Cells from spleen, mLN, siLP, and siE of single and co-infected animals were isolated and stimulated with PMA and ionomycin in the presence of Brefeldin A followed by intracellular cytokine staining. Gating strategy for the cytokines IL-5 and IFN-γ in CD4 + cells isolated from spleen (A) . Bar graphs showing frequencies of CD4 + T cells expressing IL-4 (B) , IL-5 (C) , IL-13 (D) , and IFN-γ (E) in spleen, mLN, siLP, and siE. (F) IL-4 and IFN-γ production detected by ELISA in supernatant from 3 × 10 5 splenocytes stimulated with (black bars) and without (white bars) anti-CD3/CD28 antibodies. (B–F) Data shown as mean ± SEM, pooled from two independent experiments with n = 8–9 Statistical analysis was performed using the Kruskal-Wallis with Dunn's multiple comparison test, * P ≤ 0.05, ** P ≤ 0.01, and *** P ≤ 0.001.

    Techniques Used: Infection, Mouse Assay, Isolation, Staining, Expressing, Enzyme-linked Immunosorbent Assay

    6) Product Images from "The Multiprotein Exocyst Complex Is Essential for Cell Separation in Schizosaccharomyces pombe"

    Article Title: The Multiprotein Exocyst Complex Is Essential for Cell Separation in Schizosaccharomyces pombe

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.01-11-0542

    Localization of exocyst components in S. pombe . (A) Exocyst components localize to the division site and cell tips. sec8-GFP sec6 - Myc cells were stained with antibodies against GFP and Myc. In the merged micrograph, Sec8-GFP is in red, Sec6-Myc in green, and DNA in blue. Arrowheads: tip localization; 1, 4: a single ring structure; 2, 3: double ring structures. (B) Sec8-GFP appeared as two rings in late mitotic cells using confocal microscopy and 3D viewing. Top panel, cell with 0° rotation; lower panel, cell with 139° rotation. (C and D) Merged micrographs of Sec8-GFP with Sec10-Myc (C) and Exo70-Myc (D). Sec8-GFP is in red, Sec10-Myc or Exo70-Myc in green, and DNA in blue. (E) The localization of Sec10p in relation to Myo2p, an actomyosin ring marker. cdc25 - 22 cells expressing Sec10p-GFP were synchronized in G2 by incubation at 36°C for 4 h and allowed to enter mitosis by shifting to 24°C. Cells were stained with antibodies against GFP and Myo2p. (F) The localization of Sec10p is dependent on intact F-actin structures. cdc25 - 22 cells expressing Sec10p-GFP weretreated with either LatA in DMSO (bottom panels) or DMSO alone (top panels) upon release into mitosis. Samples were stained with antibodies against GFP and α-tubulin to visualize Sec10-GFP and microtubules. (G and H) Localization of Sec10p to the division site is lost in cdc12-112 (G) and cdc15-140 (H) mutants. Cells were grown at 24°C, shifted to 36°C for 4 h, and stained with antibodies against GFP and α-tubulin to visualize Sec10-GFP and microtubules. (I) The localization of Sec10p is insensitive to brefeldin A treatment. cdc25 - 22 cells expressing Sec10-GFP or Gma12-GFP (as a marker for the Golgi) were treated with either brefeldin A in ethanol (bottom panels) or ethanol alone (top panels) upon release into mitosis. Samples were stained with DAPI and with antibodies against GFP to visualize Sec10-GFP or Gma12-GFP and DNA. Bars: (A–D) 10 μm; (E–I) 5 μm.
    Figure Legend Snippet: Localization of exocyst components in S. pombe . (A) Exocyst components localize to the division site and cell tips. sec8-GFP sec6 - Myc cells were stained with antibodies against GFP and Myc. In the merged micrograph, Sec8-GFP is in red, Sec6-Myc in green, and DNA in blue. Arrowheads: tip localization; 1, 4: a single ring structure; 2, 3: double ring structures. (B) Sec8-GFP appeared as two rings in late mitotic cells using confocal microscopy and 3D viewing. Top panel, cell with 0° rotation; lower panel, cell with 139° rotation. (C and D) Merged micrographs of Sec8-GFP with Sec10-Myc (C) and Exo70-Myc (D). Sec8-GFP is in red, Sec10-Myc or Exo70-Myc in green, and DNA in blue. (E) The localization of Sec10p in relation to Myo2p, an actomyosin ring marker. cdc25 - 22 cells expressing Sec10p-GFP were synchronized in G2 by incubation at 36°C for 4 h and allowed to enter mitosis by shifting to 24°C. Cells were stained with antibodies against GFP and Myo2p. (F) The localization of Sec10p is dependent on intact F-actin structures. cdc25 - 22 cells expressing Sec10p-GFP weretreated with either LatA in DMSO (bottom panels) or DMSO alone (top panels) upon release into mitosis. Samples were stained with antibodies against GFP and α-tubulin to visualize Sec10-GFP and microtubules. (G and H) Localization of Sec10p to the division site is lost in cdc12-112 (G) and cdc15-140 (H) mutants. Cells were grown at 24°C, shifted to 36°C for 4 h, and stained with antibodies against GFP and α-tubulin to visualize Sec10-GFP and microtubules. (I) The localization of Sec10p is insensitive to brefeldin A treatment. cdc25 - 22 cells expressing Sec10-GFP or Gma12-GFP (as a marker for the Golgi) were treated with either brefeldin A in ethanol (bottom panels) or ethanol alone (top panels) upon release into mitosis. Samples were stained with DAPI and with antibodies against GFP to visualize Sec10-GFP or Gma12-GFP and DNA. Bars: (A–D) 10 μm; (E–I) 5 μm.

    Techniques Used: Staining, Confocal Microscopy, Marker, Expressing, Incubation

    7) Product Images from "Pulmonary Group 2 Innate Lymphoid Cell Phenotype Is Context Specific: Determining the Effect of Strain, Location, and Stimuli"

    Article Title: Pulmonary Group 2 Innate Lymphoid Cell Phenotype Is Context Specific: Determining the Effect of Strain, Location, and Stimuli

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2019.03114

    ILC2 marker expression varies with ex vivo re-stimulation. (A) tSNE plot of 4,437 lung Gata3 + cells from both C57BL/6 and BALB/c mice. Representative histograms for extracellular and intracellular markers on Gata3 + cells taken from C57BL/6 and BALB/c mice either left in media or stimulated with brefeldin A (Bref) or PMA, ionomycin and Bref (PIB). Percentage of Gata3 + cells expressing (B) CD25, (C) CD90, (D) CD127, (E) KLRG1, (F) SCA1, (G) ST2, (H) IL-5, and (I) IL-13 and accompanying tSNE plots. Data is the combination of two individual experiments, n = 10 per group. †† P
    Figure Legend Snippet: ILC2 marker expression varies with ex vivo re-stimulation. (A) tSNE plot of 4,437 lung Gata3 + cells from both C57BL/6 and BALB/c mice. Representative histograms for extracellular and intracellular markers on Gata3 + cells taken from C57BL/6 and BALB/c mice either left in media or stimulated with brefeldin A (Bref) or PMA, ionomycin and Bref (PIB). Percentage of Gata3 + cells expressing (B) CD25, (C) CD90, (D) CD127, (E) KLRG1, (F) SCA1, (G) ST2, (H) IL-5, and (I) IL-13 and accompanying tSNE plots. Data is the combination of two individual experiments, n = 10 per group. †† P

    Techniques Used: Marker, Expressing, Ex Vivo, Mouse Assay

    8) Product Images from "Suppression of Autoimmune Retinal Inflammation by an Antiangiogenic Drug"

    Article Title: Suppression of Autoimmune Retinal Inflammation by an Antiangiogenic Drug

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0066219

    Lodamin inhibits Th1/Th17 cell differentiation by inhibiting cell proliferation and reducing the production of proinflammatory cytokines. (A) Purified CD4+CD25- T cells from spleen were activated by anti-CD3 antibody for 3 days under Th1 or Th17 polarization conditions in the presence or absence of Lodamin (1 nM), followed by stimulation with PMA and ionomycin in the presence of Brefeldin A; all plots are gated on CD4+ cells. Polarization conditions: Th0; anti-IFN-g Ab (10ug/ml) and anti-IL-4 Ab (10ug/ml); Th1; IL-12 (10 ng/ml) and anti-IL-4 Ab (10 µg/ml); Th17; IL-6 (20 ng/ml), TGFβ (2 ng/ml), anti-IFNγ Ab (10 µg/ml) and anti-IL-4 Ab (10 µg/ml) (B) Lodamin inhibits hallmark cytokine of Th1/Th17 under each polarization condition. IFN-γ and IL-17 protein level as measured by ELISA from activated cells supernatants (as describe in panel A). (mean ± SEM,  n = 3 , *** p
    Figure Legend Snippet: Lodamin inhibits Th1/Th17 cell differentiation by inhibiting cell proliferation and reducing the production of proinflammatory cytokines. (A) Purified CD4+CD25- T cells from spleen were activated by anti-CD3 antibody for 3 days under Th1 or Th17 polarization conditions in the presence or absence of Lodamin (1 nM), followed by stimulation with PMA and ionomycin in the presence of Brefeldin A; all plots are gated on CD4+ cells. Polarization conditions: Th0; anti-IFN-g Ab (10ug/ml) and anti-IL-4 Ab (10ug/ml); Th1; IL-12 (10 ng/ml) and anti-IL-4 Ab (10 µg/ml); Th17; IL-6 (20 ng/ml), TGFβ (2 ng/ml), anti-IFNγ Ab (10 µg/ml) and anti-IL-4 Ab (10 µg/ml) (B) Lodamin inhibits hallmark cytokine of Th1/Th17 under each polarization condition. IFN-γ and IL-17 protein level as measured by ELISA from activated cells supernatants (as describe in panel A). (mean ± SEM, n = 3 , *** p

    Techniques Used: Cell Differentiation, Purification, Enzyme-linked Immunosorbent Assay

    Lodamin inhibits Th1/Th17 cell differentiation. Whole splenocytes were activated by anti-CD3 antibody for 3 days under Th1 or Th17 polarization conditions in the presence or absence of Lodamin (vehicle, 1 nM and 10 nM), followed by stimulation with PMA and ionomycin in the presence of Brefeldin A; Cell were analyzed by FACS following intracellular cytokine staining of IL-17 and IFN-γ, all plots are gated on CD4+ cells. Data are representative of three independent results.
    Figure Legend Snippet: Lodamin inhibits Th1/Th17 cell differentiation. Whole splenocytes were activated by anti-CD3 antibody for 3 days under Th1 or Th17 polarization conditions in the presence or absence of Lodamin (vehicle, 1 nM and 10 nM), followed by stimulation with PMA and ionomycin in the presence of Brefeldin A; Cell were analyzed by FACS following intracellular cytokine staining of IL-17 and IFN-γ, all plots are gated on CD4+ cells. Data are representative of three independent results.

    Techniques Used: Cell Differentiation, FACS, Staining

    9) Product Images from "Regulation of human Th9 differentiation by type I interferons and IL-21"

    Article Title: Regulation of human Th9 differentiation by type I interferons and IL-21

    Journal: Immunology and cell biology

    doi: 10.1038/icb.2010.53

    Characterization of IL-9 production in memory CD4 + T cells. ( a ) Freshly isolated memory CD4 + T cells were stimulated with PMA and ionomycin in the presence of Brefeldin A (BFA) for 4 h and stained for intracellular expression of IL-4, IL-5, IL-13 and
    Figure Legend Snippet: Characterization of IL-9 production in memory CD4 + T cells. ( a ) Freshly isolated memory CD4 + T cells were stimulated with PMA and ionomycin in the presence of Brefeldin A (BFA) for 4 h and stained for intracellular expression of IL-4, IL-5, IL-13 and

    Techniques Used: Isolation, Staining, Expressing

    10) Product Images from "Oral Vaccination with Lipid-Formulated BCG Induces a Long-lived, Multifunctional CD4+ T Cell Memory Immune Response"

    Article Title: Oral Vaccination with Lipid-Formulated BCG Induces a Long-lived, Multifunctional CD4+ T Cell Memory Immune Response

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0045888

    Oral vaccination with Liporale™-BCG induces CD4 + T cell cytokine production in the lung. Lymphocytes from the lungs of naïve, Liporale™-BCG vaccinated (BCG oral) or subcutaneous vaccinated (BCG s.c.) mice were stimulated for 6 hours in vitro in the presence of Brefeldin A and monensin then analyzed by flow cytometry. (A) Representative plots show CD4 + T cells from the lungs of naïve or BCG vaccinated mice expressing IFNγ, TNFα or IL-2. (B) Bar graphs show the percentage of CD4 + T cells from the lungs of naïve or BCG vaccinated mice expressing cytokines at 4, 8 or 30 weeks post immunization. Results are displayed as mean + SEM of n = 5 for each group, significance expressed relative to naïve: *p
    Figure Legend Snippet: Oral vaccination with Liporale™-BCG induces CD4 + T cell cytokine production in the lung. Lymphocytes from the lungs of naïve, Liporale™-BCG vaccinated (BCG oral) or subcutaneous vaccinated (BCG s.c.) mice were stimulated for 6 hours in vitro in the presence of Brefeldin A and monensin then analyzed by flow cytometry. (A) Representative plots show CD4 + T cells from the lungs of naïve or BCG vaccinated mice expressing IFNγ, TNFα or IL-2. (B) Bar graphs show the percentage of CD4 + T cells from the lungs of naïve or BCG vaccinated mice expressing cytokines at 4, 8 or 30 weeks post immunization. Results are displayed as mean + SEM of n = 5 for each group, significance expressed relative to naïve: *p

    Techniques Used: Mouse Assay, In Vitro, Flow Cytometry, Cytometry, Expressing

    Increased multifunctional CD4 + T cells in the lungs of Liporale™-BCG vaccinated mice. Lymphocytes from the lungs of naïve, Liporale™-BCG vaccinated (BCG oral) or subcutaneous vaccinated (BCG s.c.) mice were stimulated for 6 hours in the presence of Brefeldin A and monensin then analyzed by flow cytometry. (A) Representative dot plots of CD4 + T cells from the lungs of naïve or BCG vaccinated mice expressing IFNγ, TNFα or IL-2 cytokines. Multifunctional cells were identified by first gating on IFNγ+ cells, and then on cells positive for both TNFα and IL-2. (B) Bar graph showing the percentage of CD4 + T cells from naïve or BCG vaccinated mice producing 1, 2 or 3 cytokines simultaneously, and bar graph of the frequency of multifunctional CD4 + T cells in the lungs of naïve or vaccinated mice at 4 weeks post immunization. (C,D) Bar graphs as in (B), but showing results for 8 and 30 weeks post vaccination, respectively. Results are displayed as mean + SEM of n = 5 for each group, significance expressed relative to naïve: *p
    Figure Legend Snippet: Increased multifunctional CD4 + T cells in the lungs of Liporale™-BCG vaccinated mice. Lymphocytes from the lungs of naïve, Liporale™-BCG vaccinated (BCG oral) or subcutaneous vaccinated (BCG s.c.) mice were stimulated for 6 hours in the presence of Brefeldin A and monensin then analyzed by flow cytometry. (A) Representative dot plots of CD4 + T cells from the lungs of naïve or BCG vaccinated mice expressing IFNγ, TNFα or IL-2 cytokines. Multifunctional cells were identified by first gating on IFNγ+ cells, and then on cells positive for both TNFα and IL-2. (B) Bar graph showing the percentage of CD4 + T cells from naïve or BCG vaccinated mice producing 1, 2 or 3 cytokines simultaneously, and bar graph of the frequency of multifunctional CD4 + T cells in the lungs of naïve or vaccinated mice at 4 weeks post immunization. (C,D) Bar graphs as in (B), but showing results for 8 and 30 weeks post vaccination, respectively. Results are displayed as mean + SEM of n = 5 for each group, significance expressed relative to naïve: *p

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

    11) Product Images from "A novel role for IL-22R1 as a driver of inflammation"

    Article Title: A novel role for IL-22R1 as a driver of inflammation

    Journal: Blood

    doi: 10.1182/blood-2010-05-285908

    ALK + ALCL cell line characterization and leukemia model amplify IL-22, which correlates with tumor burden . (A) Histogram of flow cytometric analysis of cell lines stained for IL-22R1 and pSTAT3 ( Tyr 705) antibodies. (B) Relative quantification of AHR, RORC, IL-22, IL-17F, and IL-26 by real-time polymerase chain reaction. (C) Zebra-plot of flow cytometry data showing intracellular levels of IL-22 with (Bref A +) or without (Bref A −) Brefeldin A, and PMA and ionomycin stimulation with Brefeldin A (P/I+ Bref A +) for 5 hours. (D) Kaplan-Meier survival plot of the Karpas299 leukemia-bearing SCID/NOD mice injected with PBS or HeFi-1. Serum levels of sIL-2Rα (E) and IL-22 (F) in Karpas299 leukemia-bearing SCID/NOD mice at day 21 after therapy.
    Figure Legend Snippet: ALK + ALCL cell line characterization and leukemia model amplify IL-22, which correlates with tumor burden . (A) Histogram of flow cytometric analysis of cell lines stained for IL-22R1 and pSTAT3 ( Tyr 705) antibodies. (B) Relative quantification of AHR, RORC, IL-22, IL-17F, and IL-26 by real-time polymerase chain reaction. (C) Zebra-plot of flow cytometry data showing intracellular levels of IL-22 with (Bref A +) or without (Bref A −) Brefeldin A, and PMA and ionomycin stimulation with Brefeldin A (P/I+ Bref A +) for 5 hours. (D) Kaplan-Meier survival plot of the Karpas299 leukemia-bearing SCID/NOD mice injected with PBS or HeFi-1. Serum levels of sIL-2Rα (E) and IL-22 (F) in Karpas299 leukemia-bearing SCID/NOD mice at day 21 after therapy.

    Techniques Used: Flow Cytometry, Staining, Real-time Polymerase Chain Reaction, Cytometry, Mouse Assay, Injection

    G-CSF, IL-22, and IL-17 are elevated in IL-22R1 tg mice . (A) Zebra plot showing intracellular levels of IL-17 and IL-22 in T cells in lungs when injected with Brefeldin A for 6 hours intravenously. Graphs showing levels of IL-17 and IL-22 as percentage (B) and total cellularity (C). (D) Histogram of flow cytometric analysis of splenocytes stimulated with PMA and ionomycin for 5 hours blocked with Brefeldin A and stained with CD4 and IL-17 antibodies. (E) Cytometric bead array to assay the concentrations of G-CSF in the serum of IL-22R1 (n = 5) tg founders and nontransgenic littermates (n = 5). The data are represented as the mean ± SEM. ** P
    Figure Legend Snippet: G-CSF, IL-22, and IL-17 are elevated in IL-22R1 tg mice . (A) Zebra plot showing intracellular levels of IL-17 and IL-22 in T cells in lungs when injected with Brefeldin A for 6 hours intravenously. Graphs showing levels of IL-17 and IL-22 as percentage (B) and total cellularity (C). (D) Histogram of flow cytometric analysis of splenocytes stimulated with PMA and ionomycin for 5 hours blocked with Brefeldin A and stained with CD4 and IL-17 antibodies. (E) Cytometric bead array to assay the concentrations of G-CSF in the serum of IL-22R1 (n = 5) tg founders and nontransgenic littermates (n = 5). The data are represented as the mean ± SEM. ** P

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

    12) Product Images from "Neuroinvasive Listeria monocytogenes Infection Triggers IFN-Activation of Microglia and Upregulates Microglial miR-155"

    Article Title: Neuroinvasive Listeria monocytogenes Infection Triggers IFN-Activation of Microglia and Upregulates Microglial miR-155

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.02751

    IFN-γ-producing CD45 high cells are recruited to brains of infected mice. (A) Heat map shows the top 5 upstream regulators of mRNA expression identified by IPA® Pathways Analysis in microglia from infected B6.WT and B6.miR-155 −/− mice. (B) CD45 high CD11b pos cells from brains of B6.WT ( ) and B6.miR-155 −/− ( ) mice 7 d p.i. described in Figure 2 were collected by FACS sorting and mRNA transcripts were quantified by nCounter. Data presented are the mean + SD nCounts of the indicated transcripts from 3 to 4 cell pools, each derived from 2 to 3 mice/genotype. Significant p -values between genotypes calculated by 2-tailed Student's t -test are given. (C–G) Brain leukocytes were harvested at 7 d p.i. from B6.WT and B6.miR-155 −/− mice infected with 4.7 × 10 5 CFU L. monocytogenes EGD then were incubated for 4 h with PMA/ionomycin plus brefeldin A and monensin. After immunolabeling of extracellular markers, the cells were fixed and permeabilized then incubated with anti-IFN-γ or isotype control mAb and analyzed by flow cytometry. (C) Dotplot shows gating of CD45 high CD11b neg/low cells from singlets. (D,E) Representative B6.WT dotplots show IFN-γ isotype (upper panels) and anti-IFN-γ (lower panels) mAbs vs. CD11b (D) and Ly6C (E) on gated CD45 high CD11b neg/low cells. Graphs (F,G) show percentages of IFN-γ pos cells from B6.WT ( ) and B6.miR-155 −/− ( ) mice from 2 separate experiments that are grouped according to expression of CD11b (F) and Ly6C (G) . Line represents the group mean. Significant p -values calculated by 2-tailed Student's t -test are given.
    Figure Legend Snippet: IFN-γ-producing CD45 high cells are recruited to brains of infected mice. (A) Heat map shows the top 5 upstream regulators of mRNA expression identified by IPA® Pathways Analysis in microglia from infected B6.WT and B6.miR-155 −/− mice. (B) CD45 high CD11b pos cells from brains of B6.WT ( ) and B6.miR-155 −/− ( ) mice 7 d p.i. described in Figure 2 were collected by FACS sorting and mRNA transcripts were quantified by nCounter. Data presented are the mean + SD nCounts of the indicated transcripts from 3 to 4 cell pools, each derived from 2 to 3 mice/genotype. Significant p -values between genotypes calculated by 2-tailed Student's t -test are given. (C–G) Brain leukocytes were harvested at 7 d p.i. from B6.WT and B6.miR-155 −/− mice infected with 4.7 × 10 5 CFU L. monocytogenes EGD then were incubated for 4 h with PMA/ionomycin plus brefeldin A and monensin. After immunolabeling of extracellular markers, the cells were fixed and permeabilized then incubated with anti-IFN-γ or isotype control mAb and analyzed by flow cytometry. (C) Dotplot shows gating of CD45 high CD11b neg/low cells from singlets. (D,E) Representative B6.WT dotplots show IFN-γ isotype (upper panels) and anti-IFN-γ (lower panels) mAbs vs. CD11b (D) and Ly6C (E) on gated CD45 high CD11b neg/low cells. Graphs (F,G) show percentages of IFN-γ pos cells from B6.WT ( ) and B6.miR-155 −/− ( ) mice from 2 separate experiments that are grouped according to expression of CD11b (F) and Ly6C (G) . Line represents the group mean. Significant p -values calculated by 2-tailed Student's t -test are given.

    Techniques Used: Infection, Mouse Assay, Expressing, Indirect Immunoperoxidase Assay, FACS, Derivative Assay, Incubation, Immunolabeling, Flow Cytometry

    miR-155 inhibits production of pro-inflammatory cytokines after in vitro stimulation with heat-killed L. monocytogenes . (A) CD11b pos brain cells (2.5 × 10 5 /ml) were collected by immunomagnetic sorting from uninfected B6.WT ( ) and B6.miR-155 −/− ( ) mice and after i.p. infection with 2.1 × 10 5 CFU L. monocytogenes strain EGD, then were incubated overnight with heat-killed L. monocytogenes EGD. Mediator concentrations in cell supernatants were measured by bead array and are presented are the mean ± SD concentration (pg/ml) from 4 mice in each genotype/time point. (B–D) Brain leukocytes from uninfected mice and from mice infected with 2.1–3.2 × 10 5 CFU L. monocytogenes strain EGD were incubated overnight with heat-killed L. monocytogenes EGD, plus Brefeldin A for the last 4–6 h. After immunolabeling of extracellular markers, the cells were fixed and permeabilized then incubated with isotype control or anti-TNF mAb and analyzed by flow cytometry. Microglia were identified as CD45 int CD11b pos cells. TNF pos microglia were identified in each experiment based on gating with isotype control mAb. Dotplots show TNF expression from representative samples including isotype (B) , and 7 d p.i. B6.WT and B6.miR-155 −/− (C) . Percentages of TNF pos microglia from uninfected mice or mice 3 d and 7 d p.i. were normalized to the mean percentage of cytokine pos microglia in B6.WT mice for that experiment and time post-infection. (D) Results from 4 separate experiments are combined and presented as the normalized % TNF pos microglia from uninfected and infected B6.WT ( ) and B6.miR-155 −/− ( ) mice, n = 8–13 mice from each genotype/time point. Symbols represent individual mice with the horizontal bar at the group mean. Significant p -values between genotypes were calculated by 2-tailed Student's t -test are given.
    Figure Legend Snippet: miR-155 inhibits production of pro-inflammatory cytokines after in vitro stimulation with heat-killed L. monocytogenes . (A) CD11b pos brain cells (2.5 × 10 5 /ml) were collected by immunomagnetic sorting from uninfected B6.WT ( ) and B6.miR-155 −/− ( ) mice and after i.p. infection with 2.1 × 10 5 CFU L. monocytogenes strain EGD, then were incubated overnight with heat-killed L. monocytogenes EGD. Mediator concentrations in cell supernatants were measured by bead array and are presented are the mean ± SD concentration (pg/ml) from 4 mice in each genotype/time point. (B–D) Brain leukocytes from uninfected mice and from mice infected with 2.1–3.2 × 10 5 CFU L. monocytogenes strain EGD were incubated overnight with heat-killed L. monocytogenes EGD, plus Brefeldin A for the last 4–6 h. After immunolabeling of extracellular markers, the cells were fixed and permeabilized then incubated with isotype control or anti-TNF mAb and analyzed by flow cytometry. Microglia were identified as CD45 int CD11b pos cells. TNF pos microglia were identified in each experiment based on gating with isotype control mAb. Dotplots show TNF expression from representative samples including isotype (B) , and 7 d p.i. B6.WT and B6.miR-155 −/− (C) . Percentages of TNF pos microglia from uninfected mice or mice 3 d and 7 d p.i. were normalized to the mean percentage of cytokine pos microglia in B6.WT mice for that experiment and time post-infection. (D) Results from 4 separate experiments are combined and presented as the normalized % TNF pos microglia from uninfected and infected B6.WT ( ) and B6.miR-155 −/− ( ) mice, n = 8–13 mice from each genotype/time point. Symbols represent individual mice with the horizontal bar at the group mean. Significant p -values between genotypes were calculated by 2-tailed Student's t -test are given.

    Techniques Used: In Vitro, Mouse Assay, Infection, Incubation, Concentration Assay, Immunolabeling, Flow Cytometry, Expressing

    13) Product Images from "Effects of Mycobacteria Major Secretion Protein, Ag85B, on Allergic Inflammation in the Lung"

    Article Title: Effects of Mycobacteria Major Secretion Protein, Ag85B, on Allergic Inflammation in the Lung

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0106807

    IFN-γ and IL-17-producing CD4-negative cell subsets proliferated in BAL fluid after rAg85B administration. OVA-immunized (i.p., day0 and 14) and sensitized (5% aerosolized-OVA, day21 to 25) BALB/c mice were challenged with PBS or rAg85B protein (i.p. (100 µg; days 0 and 14) and i.n. (20 µg; days 21, 23, and 25)). At 24 h after the last OVA sensitization, BAL fluid from naïve or OVA sensitized BALB/c mice treated with PBS or rAg85B, were harvested. BAL cells were stimulated with ionomycin and PMA for 5 h, and with brefeldin A added in the last 3 h. Flow cytometry of stimulated BAL cells from PBS-treated (upper) and rAg85B protein-treated (lower) OVA-sensitized mice stained with specific antibodies indicated marker. Numbers in quadrants indicate percent of cells in each ( A ). Absolute numbers of various cell populations (above graphs) in BAL fluid ( B , C ). Data are representative of three independent experiments (**P
    Figure Legend Snippet: IFN-γ and IL-17-producing CD4-negative cell subsets proliferated in BAL fluid after rAg85B administration. OVA-immunized (i.p., day0 and 14) and sensitized (5% aerosolized-OVA, day21 to 25) BALB/c mice were challenged with PBS or rAg85B protein (i.p. (100 µg; days 0 and 14) and i.n. (20 µg; days 21, 23, and 25)). At 24 h after the last OVA sensitization, BAL fluid from naïve or OVA sensitized BALB/c mice treated with PBS or rAg85B, were harvested. BAL cells were stimulated with ionomycin and PMA for 5 h, and with brefeldin A added in the last 3 h. Flow cytometry of stimulated BAL cells from PBS-treated (upper) and rAg85B protein-treated (lower) OVA-sensitized mice stained with specific antibodies indicated marker. Numbers in quadrants indicate percent of cells in each ( A ). Absolute numbers of various cell populations (above graphs) in BAL fluid ( B , C ). Data are representative of three independent experiments (**P

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

    IFN-γ and IL-17-producing CD4 T cell subsets proliferated in lymph nodes after rAg85B administration. OVA-immunized (i.p., day0 and 14) and sensitized (5% aerosolized-OVA, day21 to 25) BALB/c mice were challenged with PBS or rAg85B protein (i.p. (100 µg; days 0 and 14) and i.n. (20 µg; days 21, 23, and 25)). At 24 h after the last OVA sensitization, mediastinal lymph nodes (MLNs) from naïve or OVA sensitized BALB/c mice treated with PBS or rAg85B, were harvested. MLNs were stimulated with ionomycin and PMA for 5 h, and with brefeldin A added in the last 3 h. Flow cytometry of stimulated MLNs from naïve (upper), PBS-treated (middle) and rAg85B protein-treated (lower) OVA-sensitized mice stained with specific antibodies indicated marker. Numbers in quadrants indicate percent of cells in each ( A ). Absolute numbers of various cell populations (above graphs) in lymph nodes ( B , C ). Data are representative of three independent experiments (*P
    Figure Legend Snippet: IFN-γ and IL-17-producing CD4 T cell subsets proliferated in lymph nodes after rAg85B administration. OVA-immunized (i.p., day0 and 14) and sensitized (5% aerosolized-OVA, day21 to 25) BALB/c mice were challenged with PBS or rAg85B protein (i.p. (100 µg; days 0 and 14) and i.n. (20 µg; days 21, 23, and 25)). At 24 h after the last OVA sensitization, mediastinal lymph nodes (MLNs) from naïve or OVA sensitized BALB/c mice treated with PBS or rAg85B, were harvested. MLNs were stimulated with ionomycin and PMA for 5 h, and with brefeldin A added in the last 3 h. Flow cytometry of stimulated MLNs from naïve (upper), PBS-treated (middle) and rAg85B protein-treated (lower) OVA-sensitized mice stained with specific antibodies indicated marker. Numbers in quadrants indicate percent of cells in each ( A ). Absolute numbers of various cell populations (above graphs) in lymph nodes ( B , C ). Data are representative of three independent experiments (*P

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

    Innate immune cells that secrete Th17-related cytokines are induced by rAg85B administration in BAL fluid. OVA-immunized (i.p., day0 and 14) and sensitized (5% aerosolized-OVA, day21 to 25) BALB/c mice were challenged with PBS or rAg85B protein (i.p. (100 µg; days 0 and 14) and i.n. (20 µg; days 21, 23, and 25)). At 24 h after the last OVA sensitization, BAL fluid from naïve or OVA sensitized BALB/c mice treated with PBS or rAg85B, were harvested. BAL cells were stimulated with ionomycin and PMA for 5 h, and with brefeldin A added in the last 3 h. Flow cytometry of BAL cells from PBS-treated (upper) and rAg85B protein-treated (lower) OVA-sensitized mice stained with anti-CD3, anti-CD4, anti-CD8, anti-Gr-1, anti-γδ TCR, anti-NKp46, anti-CD11c, anti-CD127 (IL-7R) and Lineage specific marker (CD3, CD19, Gr-1, CD11b, CD11c). Numbers in quadrants indicate percent of cells in each ( A ). Intracellular IL-17 and IL-22 staining in indicated cells by flow cytometry (dot plots) and absolute numbers of those cell populations (side graphs) in the BAL fluid ( B, C, D, E, F, G ). Data are representative of at least two independent experiments (**P
    Figure Legend Snippet: Innate immune cells that secrete Th17-related cytokines are induced by rAg85B administration in BAL fluid. OVA-immunized (i.p., day0 and 14) and sensitized (5% aerosolized-OVA, day21 to 25) BALB/c mice were challenged with PBS or rAg85B protein (i.p. (100 µg; days 0 and 14) and i.n. (20 µg; days 21, 23, and 25)). At 24 h after the last OVA sensitization, BAL fluid from naïve or OVA sensitized BALB/c mice treated with PBS or rAg85B, were harvested. BAL cells were stimulated with ionomycin and PMA for 5 h, and with brefeldin A added in the last 3 h. Flow cytometry of BAL cells from PBS-treated (upper) and rAg85B protein-treated (lower) OVA-sensitized mice stained with anti-CD3, anti-CD4, anti-CD8, anti-Gr-1, anti-γδ TCR, anti-NKp46, anti-CD11c, anti-CD127 (IL-7R) and Lineage specific marker (CD3, CD19, Gr-1, CD11b, CD11c). Numbers in quadrants indicate percent of cells in each ( A ). Intracellular IL-17 and IL-22 staining in indicated cells by flow cytometry (dot plots) and absolute numbers of those cell populations (side graphs) in the BAL fluid ( B, C, D, E, F, G ). Data are representative of at least two independent experiments (**P

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

    14) Product Images from "Weakly acidic pH reduces inflammatory cytokine expression in airway epithelial cells"

    Article Title: Weakly acidic pH reduces inflammatory cytokine expression in airway epithelial cells

    Journal: Respiratory Research

    doi: 10.1186/s12931-016-0399-3

    Inflammatory cytokine expression in AECs stimulated with LPS in weakly acidic media for 4 h. AECs were stimulated with 5 μg/ml LPS in media adjusted to pH6.5 – pH5.5 with HCl for 4 h ( n = 3). Normal, unadjusted BEGM is pH7.4; this was used as a control to show basal IL-6 and IL-8 expression. LPS stimulation at pH7.4 was used as a control for normal AEC response to LPS. Brefeldin A (BA) was used with LPS as a positive control for intracellular protein retention. a IL-6 and IL-8 mRNA expression was measured by qPCR. Fold change is related to pH7.4 control. b Cytosolic IL-6 and IL-8 protein was measured in pg/ml by ELISA of whole cell lysates and was normalised to total protein concentration, measured in μg/ml by BCA assay. Cytosolic cytokine was calculated in ng. c Secretion of IL-6 and IL-8 proteins was measured in pg/ml by ELISA of cell supernatants. d Cell viability was measured by MTT assay. ( n = 3) All test pH conditions were compared to pH7.4 + LPS for statistical analysis. Values presented are mean ± SEM * p
    Figure Legend Snippet: Inflammatory cytokine expression in AECs stimulated with LPS in weakly acidic media for 4 h. AECs were stimulated with 5 μg/ml LPS in media adjusted to pH6.5 – pH5.5 with HCl for 4 h ( n = 3). Normal, unadjusted BEGM is pH7.4; this was used as a control to show basal IL-6 and IL-8 expression. LPS stimulation at pH7.4 was used as a control for normal AEC response to LPS. Brefeldin A (BA) was used with LPS as a positive control for intracellular protein retention. a IL-6 and IL-8 mRNA expression was measured by qPCR. Fold change is related to pH7.4 control. b Cytosolic IL-6 and IL-8 protein was measured in pg/ml by ELISA of whole cell lysates and was normalised to total protein concentration, measured in μg/ml by BCA assay. Cytosolic cytokine was calculated in ng. c Secretion of IL-6 and IL-8 proteins was measured in pg/ml by ELISA of cell supernatants. d Cell viability was measured by MTT assay. ( n = 3) All test pH conditions were compared to pH7.4 + LPS for statistical analysis. Values presented are mean ± SEM * p

    Techniques Used: Expressing, Positive Control, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Protein Concentration, BIA-KA, MTT Assay

    Inflammatory cytokine expression in AECs stimulated with LPS in weakly acidic media for 16 h. AECs were stimulated with 5 μg/ml LPS in media adjusted to pH6.5 – pH5.5 with HCl for 16 h ( n = 3). Normal, unadjusted BEGM is pH7.4; this was used as a control to show basal IL-6 and IL-8 expression. LPS stimulation at pH7.4 was used as a control for normal AEC response to LPS. Brefeldin A (BA) was used with LPS as a positive control for intracellular protein retention. a IL-6 and IL-8 mRNA expression was measured by qPCR. Fold change is related to pH7.4 control. b Cytosolic IL-6 and IL-8 protein was measured in pg/ml by ELISA of whole cell lysates and was normalised to total protein concentration, measured in μg/ml by BCA assay. Cytosolic cytokine was calculated in ng. c Secretion of IL-6 and IL-8 proteins was measured in pg/ml by ELISA of cell supernatants. d Cell viability was measured by MTT assay. ( n = 3) All test pH conditions were compared to pH7.4 + LPS for statistical analysis. Values presented are mean ± SEM * p
    Figure Legend Snippet: Inflammatory cytokine expression in AECs stimulated with LPS in weakly acidic media for 16 h. AECs were stimulated with 5 μg/ml LPS in media adjusted to pH6.5 – pH5.5 with HCl for 16 h ( n = 3). Normal, unadjusted BEGM is pH7.4; this was used as a control to show basal IL-6 and IL-8 expression. LPS stimulation at pH7.4 was used as a control for normal AEC response to LPS. Brefeldin A (BA) was used with LPS as a positive control for intracellular protein retention. a IL-6 and IL-8 mRNA expression was measured by qPCR. Fold change is related to pH7.4 control. b Cytosolic IL-6 and IL-8 protein was measured in pg/ml by ELISA of whole cell lysates and was normalised to total protein concentration, measured in μg/ml by BCA assay. Cytosolic cytokine was calculated in ng. c Secretion of IL-6 and IL-8 proteins was measured in pg/ml by ELISA of cell supernatants. d Cell viability was measured by MTT assay. ( n = 3) All test pH conditions were compared to pH7.4 + LPS for statistical analysis. Values presented are mean ± SEM * p

    Techniques Used: Expressing, Positive Control, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Protein Concentration, BIA-KA, MTT Assay

    15) Product Images from "Antibacterial autophagy occurs at PtdIns(3)P-enriched domains of the endoplasmic reticulum and requires Rab1 GTPase"

    Article Title: Antibacterial autophagy occurs at PtdIns(3)P-enriched domains of the endoplasmic reticulum and requires Rab1 GTPase

    Journal: Autophagy

    doi: 10.4161/auto.7.1.13840

    Rab1 is involved in autophagy independent of its role in ER-to-Golgi transport. (A) HeLa cells were transfected with GFP-LC3 and treated with rapamycin for 2 h. Where indicated, cells were also treated with Brefeldin A (BFA), WTM or 3-MA. The number of
    Figure Legend Snippet: Rab1 is involved in autophagy independent of its role in ER-to-Golgi transport. (A) HeLa cells were transfected with GFP-LC3 and treated with rapamycin for 2 h. Where indicated, cells were also treated with Brefeldin A (BFA), WTM or 3-MA. The number of

    Techniques Used: Transfection

    16) Product Images from "Pim1 permits generation and survival of CD4+ T cells in the absence of γc cytokine receptor signaling"

    Article Title: Pim1 permits generation and survival of CD4+ T cells in the absence of γc cytokine receptor signaling

    Journal: European journal of immunology

    doi: 10.1002/eji.201242686

    (A) Thymocyte profiles of WT and Pim1 Tg mice. Contour plots show CD4/CD8 profiles of total (top) and TCRβ + gated thymocytes (bottom). Gating strategy is shown in histograms (middle). Numbers indicate percentages of gated cells. Data shown are representative of eight independent experiments. (B) Thymocyte numbers in WT and Pim1 Tg mice. Data are shown as mean +/− SEM of 11 WT and 8 Pim1 Tg mice. (C) DN thymocyte differentiation. Lineage marker negative (Lin − ) DN thymocytes were assessed for DN1–DN4 differentiation by CD44/CD25 expression. (D) Thymocyte selection in WT and Pim1 Tg mice. Whole thymocytes were stained for CD69 and TCRβ expression. Data shown are representative of eight independent experiments. (E) LN cell numbers of WT and Pim1 Tg mice. Data are shown as mean +/− SEM of 8 WT and 7 Pim1 Tg mice. (F) LN cell profiles of WT and Pim1 Tg mice. CD4/CD8 contour plots are representative of eight independent experiments. (G) Cytokine expression in CD4 + LN T-cells. Freshly isolated CD4 + T-cells were stimulated for 3 hours with PMA + ionomycin in the presence of brefeldin A and assessed for intracellular IL-17 and IFN-γ expression. Data are representative of three independent experiments. (H) T-cell adoptive transfer into WT or Pim1 Tg hosts. WT (CD45.1) T-cells labeled with the CFSE analog “CellTrace™ Violet” were tail vein injected and analyzed 6 days later from LN of WT (CD45.2) or Pim1 Tg (CD45.2) host mice. Data shown are representative of two experiments. (B, D) Data shown are from one experiment representative of eight experiments performed. *p
    Figure Legend Snippet: (A) Thymocyte profiles of WT and Pim1 Tg mice. Contour plots show CD4/CD8 profiles of total (top) and TCRβ + gated thymocytes (bottom). Gating strategy is shown in histograms (middle). Numbers indicate percentages of gated cells. Data shown are representative of eight independent experiments. (B) Thymocyte numbers in WT and Pim1 Tg mice. Data are shown as mean +/− SEM of 11 WT and 8 Pim1 Tg mice. (C) DN thymocyte differentiation. Lineage marker negative (Lin − ) DN thymocytes were assessed for DN1–DN4 differentiation by CD44/CD25 expression. (D) Thymocyte selection in WT and Pim1 Tg mice. Whole thymocytes were stained for CD69 and TCRβ expression. Data shown are representative of eight independent experiments. (E) LN cell numbers of WT and Pim1 Tg mice. Data are shown as mean +/− SEM of 8 WT and 7 Pim1 Tg mice. (F) LN cell profiles of WT and Pim1 Tg mice. CD4/CD8 contour plots are representative of eight independent experiments. (G) Cytokine expression in CD4 + LN T-cells. Freshly isolated CD4 + T-cells were stimulated for 3 hours with PMA + ionomycin in the presence of brefeldin A and assessed for intracellular IL-17 and IFN-γ expression. Data are representative of three independent experiments. (H) T-cell adoptive transfer into WT or Pim1 Tg hosts. WT (CD45.1) T-cells labeled with the CFSE analog “CellTrace™ Violet” were tail vein injected and analyzed 6 days later from LN of WT (CD45.2) or Pim1 Tg (CD45.2) host mice. Data shown are representative of two experiments. (B, D) Data shown are from one experiment representative of eight experiments performed. *p

    Techniques Used: Mouse Assay, Marker, Expressing, Selection, Staining, Isolation, Adoptive Transfer Assay, Labeling, Injection

    17) Product Images from "Treatment with Dexamethasone and Monophosphoryl Lipid A Removes Disease-Associated Transcriptional Signatures in Monocyte-Derived Dendritic Cells from Rheumatoid Arthritis Patients and Confers Tolerogenic Features"

    Article Title: Treatment with Dexamethasone and Monophosphoryl Lipid A Removes Disease-Associated Transcriptional Signatures in Monocyte-Derived Dendritic Cells from Rheumatoid Arthritis Patients and Confers Tolerogenic Features

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2016.00458

    MPLA-tDCs modulate CD4+ T cell responses to synovial antigens . Monocytes ( n = 6–9) were differentiated into mDCs and MPLA-tDCs as described in the Section “ Materials and Methods .” At day 4 of DC generation, 4 h prior to stimulation with MPLA, mDCs and MPLA-tDCs were loaded with 1 μg/ml tuberculin purified protein derivative (PPD), 200 μg/ml synovial fluid (SF), or left unloaded for further 24 h. For the assessment of their T cell-stimulatory capacity, antigen-loaded or unloaded mDCs and MPLA-tDCs were co-cultured with autologous CFSE-labeled CD4+ T cells in a ratio of 1:2 (DC:T cell) for 6 days. To detect IFN-γ intracellularly, cells were stimulated with 50 ng/ml phorbol-12-myriastate-13-acetate and 1 μg/ml ionomycin in the presence of 1 μg/ml brefeldin-A. Proliferation-associated CFSE dilution and IFN-γ production of CD4+ T cells were analyzed by flow cytometry. (A) Representative dot plots and (B) graphic representation of the percentage of IFN-γ-producing proliferating (CFSE low ) CD4+ T cells are shown. (C) IL-17A secretion was measured in supernatants of co-cultures by ELISA. (B,C) , Box plots show median, 25- and 75%-quartiles and both extreme values (* P
    Figure Legend Snippet: MPLA-tDCs modulate CD4+ T cell responses to synovial antigens . Monocytes ( n = 6–9) were differentiated into mDCs and MPLA-tDCs as described in the Section “ Materials and Methods .” At day 4 of DC generation, 4 h prior to stimulation with MPLA, mDCs and MPLA-tDCs were loaded with 1 μg/ml tuberculin purified protein derivative (PPD), 200 μg/ml synovial fluid (SF), or left unloaded for further 24 h. For the assessment of their T cell-stimulatory capacity, antigen-loaded or unloaded mDCs and MPLA-tDCs were co-cultured with autologous CFSE-labeled CD4+ T cells in a ratio of 1:2 (DC:T cell) for 6 days. To detect IFN-γ intracellularly, cells were stimulated with 50 ng/ml phorbol-12-myriastate-13-acetate and 1 μg/ml ionomycin in the presence of 1 μg/ml brefeldin-A. Proliferation-associated CFSE dilution and IFN-γ production of CD4+ T cells were analyzed by flow cytometry. (A) Representative dot plots and (B) graphic representation of the percentage of IFN-γ-producing proliferating (CFSE low ) CD4+ T cells are shown. (C) IL-17A secretion was measured in supernatants of co-cultures by ELISA. (B,C) , Box plots show median, 25- and 75%-quartiles and both extreme values (* P

    Techniques Used: Purification, Cell Culture, Labeling, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay

    18) Product Images from "Chronic thoracic spinal cord injury impairs CD8+ T-cell function by up-regulating programmed cell death-1 expression"

    Article Title: Chronic thoracic spinal cord injury impairs CD8+ T-cell function by up-regulating programmed cell death-1 expression

    Journal: Journal of Neuroinflammation

    doi: 10.1186/1742-2094-11-65

    Blocking PD-1 restores the TNF-α production by CD8 + T-cells from chronic spinal cord injury (SCI) mice. Splenocytes (1 × 10 6 ) isolated from uninjured mice were stimulated ex vivo with PMA/ionomycin in the presence of brefeldin A for four hours (group: CT). Splenocytes (1 × 10 6 ) isolated from chronic SCI mice were also stimulated ex vivo with the same condition as the CT group, except that 10 μg/mL anti-PD-1 blocking antibody (group: SCI + αPD-1) or 10 μg/mL rat IgG2a, κ isotype (group: SCI + Isotype) were added to the culture. (A) Representative dot plots show the percentage of IFN-γ + cells in gated CD4 + T-cells. (B) Bar graph represents the mean ± SEM percentage of IFN-γ + cells in CD4 + T-cells. (C) Representative dot plots show the percentage of TNF-α + cells in gated CD8 + T-cells. (D) Bar graph represents the mean ± SEM percentage of TNF-α + cells in CD8 + T-cells. Ten thousand events gated on live singlets were collected. n = 9 for CT, n = 11 for SCI + Isotype, n = 11 for SCI + αPD-1. Data are pooled across three independent experiments. * P
    Figure Legend Snippet: Blocking PD-1 restores the TNF-α production by CD8 + T-cells from chronic spinal cord injury (SCI) mice. Splenocytes (1 × 10 6 ) isolated from uninjured mice were stimulated ex vivo with PMA/ionomycin in the presence of brefeldin A for four hours (group: CT). Splenocytes (1 × 10 6 ) isolated from chronic SCI mice were also stimulated ex vivo with the same condition as the CT group, except that 10 μg/mL anti-PD-1 blocking antibody (group: SCI + αPD-1) or 10 μg/mL rat IgG2a, κ isotype (group: SCI + Isotype) were added to the culture. (A) Representative dot plots show the percentage of IFN-γ + cells in gated CD4 + T-cells. (B) Bar graph represents the mean ± SEM percentage of IFN-γ + cells in CD4 + T-cells. (C) Representative dot plots show the percentage of TNF-α + cells in gated CD8 + T-cells. (D) Bar graph represents the mean ± SEM percentage of TNF-α + cells in CD8 + T-cells. Ten thousand events gated on live singlets were collected. n = 9 for CT, n = 11 for SCI + Isotype, n = 11 for SCI + αPD-1. Data are pooled across three independent experiments. * P

    Techniques Used: Blocking Assay, Mouse Assay, Isolation, Ex Vivo

    Impaired T-cell cytokine production in response to PMA/ionomycin stimulation after chronic spinal cord injury (SCI). Isolated splenocytes (1 × 10 6 ) from uninjured (CT) or T9-SCI mice at chronic phase after injury (SCI) were stimulated ex vivo with PMA/ionomycin in the presence of brefeldin A for four hours and then processed for flow cytometry analysis. The unstimulated controls were incubated only with brefeldin A. (A) Representative dot plots show the percentage of IFN-γ + and TNF-α + cells in gated CD4 + T-cells following PMA/ionomycin stimulation compared to unstimulated or isotype control. (B) Bar graph represents the mean ± SEM percentages and numbers of cytokine producing CD4 + T-cell in response to PMA/ionomycin stimulation. (C) Representative dot plots show the percentage of IFN-γ + and TNF-α + cells in gated CD8 + T-cells following PMA/ionomycin stimulation compared to unstimulated or isotype control. (D) Bar graph represents the mean ± SEM percentages and numbers of cytokine producing CD8 + T-cells in response to PMA/ionomycin stimulation. Ten thousand events gated on live singlets were collected. n = 14 for CT, n = 12 for SCI. Data were pooled across four independent experiments. * P
    Figure Legend Snippet: Impaired T-cell cytokine production in response to PMA/ionomycin stimulation after chronic spinal cord injury (SCI). Isolated splenocytes (1 × 10 6 ) from uninjured (CT) or T9-SCI mice at chronic phase after injury (SCI) were stimulated ex vivo with PMA/ionomycin in the presence of brefeldin A for four hours and then processed for flow cytometry analysis. The unstimulated controls were incubated only with brefeldin A. (A) Representative dot plots show the percentage of IFN-γ + and TNF-α + cells in gated CD4 + T-cells following PMA/ionomycin stimulation compared to unstimulated or isotype control. (B) Bar graph represents the mean ± SEM percentages and numbers of cytokine producing CD4 + T-cell in response to PMA/ionomycin stimulation. (C) Representative dot plots show the percentage of IFN-γ + and TNF-α + cells in gated CD8 + T-cells following PMA/ionomycin stimulation compared to unstimulated or isotype control. (D) Bar graph represents the mean ± SEM percentages and numbers of cytokine producing CD8 + T-cells in response to PMA/ionomycin stimulation. Ten thousand events gated on live singlets were collected. n = 14 for CT, n = 12 for SCI. Data were pooled across four independent experiments. * P

    Techniques Used: Isolation, Mouse Assay, Ex Vivo, Flow Cytometry, Cytometry, Incubation

    Impaired T-cell cytokine production following T-cell receptor (TCR) activation in chronic spinal cord injury (SCI) mice. Isolated splenocytes (1 × 10 6 ) from uninjured (CT) or T9-SCI mice at chronic phase after injury (SCI) were stimulated ex vivo for three days with anti-CD3 + anti-CD28 + IL-2 or with IL-2 only. Brefeldin A was added six hours before cell collection. Intracellular cytokine staining and flow cytometry analysis were performed. (A) Representative dot plots show the percentage of IFN-γ + cells and TNF-α + cells in gated CD4 + T-cells following three-day stimulation with anti-CD3 + anti-CD28 + IL-2 or with IL-2 only. (B) Bar graph represents the mean ± SEM percentages of cytokine producing CD4 + T-cell in response to TCR activation. (C) Representative dot plots show the percentage of IFN-γ + cells and TNF-α + cells in gated CD8 + T-cells following three-day stimulation with anti-CD3 + anti-CD28 + IL-2 or with IL-2 only. (D) Bar graph represents the mean ± SEM percentages of cytokine producing CD8 + T-cells in response to TCR activation. n = 4 for CT, n = 5 for SCI. Twenty thousand events gated on live singlets were collected for flow cytometry analysis. n = 5 mice per group. * P
    Figure Legend Snippet: Impaired T-cell cytokine production following T-cell receptor (TCR) activation in chronic spinal cord injury (SCI) mice. Isolated splenocytes (1 × 10 6 ) from uninjured (CT) or T9-SCI mice at chronic phase after injury (SCI) were stimulated ex vivo for three days with anti-CD3 + anti-CD28 + IL-2 or with IL-2 only. Brefeldin A was added six hours before cell collection. Intracellular cytokine staining and flow cytometry analysis were performed. (A) Representative dot plots show the percentage of IFN-γ + cells and TNF-α + cells in gated CD4 + T-cells following three-day stimulation with anti-CD3 + anti-CD28 + IL-2 or with IL-2 only. (B) Bar graph represents the mean ± SEM percentages of cytokine producing CD4 + T-cell in response to TCR activation. (C) Representative dot plots show the percentage of IFN-γ + cells and TNF-α + cells in gated CD8 + T-cells following three-day stimulation with anti-CD3 + anti-CD28 + IL-2 or with IL-2 only. (D) Bar graph represents the mean ± SEM percentages of cytokine producing CD8 + T-cells in response to TCR activation. n = 4 for CT, n = 5 for SCI. Twenty thousand events gated on live singlets were collected for flow cytometry analysis. n = 5 mice per group. * P

    Techniques Used: Activation Assay, Mouse Assay, Isolation, Ex Vivo, Staining, Flow Cytometry, Cytometry

    Exposure to norepinephrine (NE) in vitro impairs T-cell cytokine production in response to PMA/ionomycin stimulation. Enriched splenic T-cells (10 6 cells/ml) were cultured with 10 μM NE or its vehicle (Vehicle) in vitro . After two days of NE exposure, cells were stimulated with PMA/ionomycin for four hours in the presence of brefeldin A. Intracellular cytokine staining and flow cytometry analysis were performed to measure cytokine production. (A) Representative dot plots show the percentage of IFN-γ + cells and TNF-α + cells in gated CD4 + T-cells following PMA/ionomycin stimulation or with brefeldin A only (unstimulated). (B) Bar graph represents the mean ± SEM percentages cytokine producing CD4 + T-cell in response to PMA/ionomycin stimulation. (C) Representative dot plots show the percentage of IFN-γ + cells and TNF-α + cells in gated CD8 + T-cells following PMA/ionomycin stimulation or with brefeldin A only (unstimulated). (D) Bar graph represents the mean ± SEM percentages of cytokine producing CD8 + T-cells in response to PMA/ionomycin stimulation. Ten thousand events gated on lymphocytes were collected. Experiments were performed in triplicate, * P
    Figure Legend Snippet: Exposure to norepinephrine (NE) in vitro impairs T-cell cytokine production in response to PMA/ionomycin stimulation. Enriched splenic T-cells (10 6 cells/ml) were cultured with 10 μM NE or its vehicle (Vehicle) in vitro . After two days of NE exposure, cells were stimulated with PMA/ionomycin for four hours in the presence of brefeldin A. Intracellular cytokine staining and flow cytometry analysis were performed to measure cytokine production. (A) Representative dot plots show the percentage of IFN-γ + cells and TNF-α + cells in gated CD4 + T-cells following PMA/ionomycin stimulation or with brefeldin A only (unstimulated). (B) Bar graph represents the mean ± SEM percentages cytokine producing CD4 + T-cell in response to PMA/ionomycin stimulation. (C) Representative dot plots show the percentage of IFN-γ + cells and TNF-α + cells in gated CD8 + T-cells following PMA/ionomycin stimulation or with brefeldin A only (unstimulated). (D) Bar graph represents the mean ± SEM percentages of cytokine producing CD8 + T-cells in response to PMA/ionomycin stimulation. Ten thousand events gated on lymphocytes were collected. Experiments were performed in triplicate, * P

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

    19) Product Images from "Imaging-Based Reporter Systems to Define CVB-Induced Membrane Remodeling in Living Cells"

    Article Title: Imaging-Based Reporter Systems to Define CVB-Induced Membrane Remodeling in Living Cells

    Journal: Viruses

    doi: 10.3390/v12101074

    Time-lapse imaging of CVB infected cells expressing RepOr. ( a )  Top , the bicistronic RepOr cassette contains a Golgi localized mEmerald (mEm-Golgi) followed by an IRES-driven RepER, where the GFP has been substituted with BFP. Signal peptide (SP), transmembrane domain (TM), and nuclear localization signal (NLS).  Bottom , Schematic of the predicted model for visualizing the Golgi, ER, and enterovirus infection. In uninfected cells (left), mCherry and BFP colocalize in the ER (purple), and mEmerald is localized to the Golgi (green). During enterovirus infection (right), viral 3C pro  cleavage releases the BFP-NLS, which translocates to the nucleus, the mCherry is retained in the ER (red) and the Golgi remains (green). ( b ) Representative time-points of an image series from live-cell imaging of mEmerald (Golgi, green), BFP (reporter, gray), mCherry (ER, red), and merged panels from U2OS cells expressing RepOr treated with DMSO, brefeldin A, or infected with CVB. Scale bars are 20 μm. ( c ) Quantification of infection (blue) and Golgi area (green) in mock (dashed lines,  n  = 16) and CVB (solid lines,  n  = 16) infected cells shown as the average fold change compared to 0 hpi; the gray dashed line shows a fold change of 1. ( d ) Scatter plot of infection and Golgi areas. Transparent dots represent individual measurements taken every 15 min for individual mock (gray,  n  = 16) or CVB (green,  n  = 16) infected cells from 0 to 6 hpi, solid outlined dots represent the averages.
    Figure Legend Snippet: Time-lapse imaging of CVB infected cells expressing RepOr. ( a ) Top , the bicistronic RepOr cassette contains a Golgi localized mEmerald (mEm-Golgi) followed by an IRES-driven RepER, where the GFP has been substituted with BFP. Signal peptide (SP), transmembrane domain (TM), and nuclear localization signal (NLS). Bottom , Schematic of the predicted model for visualizing the Golgi, ER, and enterovirus infection. In uninfected cells (left), mCherry and BFP colocalize in the ER (purple), and mEmerald is localized to the Golgi (green). During enterovirus infection (right), viral 3C pro cleavage releases the BFP-NLS, which translocates to the nucleus, the mCherry is retained in the ER (red) and the Golgi remains (green). ( b ) Representative time-points of an image series from live-cell imaging of mEmerald (Golgi, green), BFP (reporter, gray), mCherry (ER, red), and merged panels from U2OS cells expressing RepOr treated with DMSO, brefeldin A, or infected with CVB. Scale bars are 20 μm. ( c ) Quantification of infection (blue) and Golgi area (green) in mock (dashed lines, n = 16) and CVB (solid lines, n = 16) infected cells shown as the average fold change compared to 0 hpi; the gray dashed line shows a fold change of 1. ( d ) Scatter plot of infection and Golgi areas. Transparent dots represent individual measurements taken every 15 min for individual mock (gray, n = 16) or CVB (green, n = 16) infected cells from 0 to 6 hpi, solid outlined dots represent the averages.

    Techniques Used: Imaging, Infection, Expressing, Live Cell Imaging

    20) Product Images from "The Long Noncoding RNA IFNG-AS1 Promotes T Helper Type 1 Cells Response in Patients with Hashimoto’s Thyroiditis"

    Article Title: The Long Noncoding RNA IFNG-AS1 Promotes T Helper Type 1 Cells Response in Patients with Hashimoto’s Thyroiditis

    Journal: Scientific Reports

    doi: 10.1038/srep17702

    Influence of IFNG-AS1 on the transcription of IFNG in vitro . Human CD4 + T cells were purified from PBMCs by magnetic beads and transfected with IFNG-AS1 -specific siRNA and negative control (100 nM) in the presence of functional anti-human CD3 mAb and anti-human CD28 mAb for 24 h to determine the transcript level of IFNG , or 48 h before restimulation with PMA, ionomycin and brefeldin-A to determine the proportion of IFN-γ + cells. ( A ) The transcript level of IFNG-AS1 was determined by qRT-PCR after transfection. ( B ) The transcript level of IFNG mRNA was determined by qRT-PCR after transfection. ( C ) The proportion of IFN-γ + cells was analyzed by flow cytometric analysis. ( D ) The transcript level of IFNG-AS1 was detected by qRT-PCR after transfection after transfection with IFNG-AS1 -specific siRNA in a dose-dependent manner. ( E ) The proportion of IFN-γ + cells was analyzed by flow cytometric analysis after transfection with IFNG-AS1 -specific siRNA in a dose-dependent manner. The results are indicated as the means ± SD of three independent experiments, horizontal lines show the mean. * p
    Figure Legend Snippet: Influence of IFNG-AS1 on the transcription of IFNG in vitro . Human CD4 + T cells were purified from PBMCs by magnetic beads and transfected with IFNG-AS1 -specific siRNA and negative control (100 nM) in the presence of functional anti-human CD3 mAb and anti-human CD28 mAb for 24 h to determine the transcript level of IFNG , or 48 h before restimulation with PMA, ionomycin and brefeldin-A to determine the proportion of IFN-γ + cells. ( A ) The transcript level of IFNG-AS1 was determined by qRT-PCR after transfection. ( B ) The transcript level of IFNG mRNA was determined by qRT-PCR after transfection. ( C ) The proportion of IFN-γ + cells was analyzed by flow cytometric analysis. ( D ) The transcript level of IFNG-AS1 was detected by qRT-PCR after transfection after transfection with IFNG-AS1 -specific siRNA in a dose-dependent manner. ( E ) The proportion of IFN-γ + cells was analyzed by flow cytometric analysis after transfection with IFNG-AS1 -specific siRNA in a dose-dependent manner. The results are indicated as the means ± SD of three independent experiments, horizontal lines show the mean. * p

    Techniques Used: In Vitro, Purification, Magnetic Beads, Transfection, Negative Control, Functional Assay, Quantitative RT-PCR, Flow Cytometry

    21) Product Images from "Helminth-Induced Production of TGFβ and Suppression of Graft-Versus-Host Disease Is Dependent on Interleukin-4 Production by Host Cells"

    Article Title: Helminth-Induced Production of TGFβ and Suppression of Graft-Versus-Host Disease Is Dependent on Interleukin-4 Production by Host Cells

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

    doi: 10.4049/jimmunol.1700638

    Helminthic induction and maintenance of TGFβ production requires IL4 production by Foxp3 − CD4 T cells. (A) TGFβ concentration in supernatants of anti-CD3/28-stimulated MLN cultures from Hpb -infected and uninfected 8–9 week old male IL4−/− or WT BALB/c mice, as measured by ELISA. Data show mean±SD from ≥3 independent experiments, with each experiment containing multiple determinations (N indicates the number of independent determinations). p value as indicated on the figure between Hpb -infected vs. uninfected groups; differences between groups determined by unpaired Welch’s t-test. (B) Anti-CD3/28 stimulated MLN T cells from Hpb -infected WT BALB/c mice, as described in Methods were cultured with anti-IL4 blocking (anti-IL4 (+)) isotype control antibodies (Isotype Control), or no antibody added (anti-IL4(–)), as indicated. Supernatants were analyzed for TGFβ content by ELISA. Data show mean±SD from a representative experiment of 5 independent experiments, with each experiment containing multiple determinations; p values as shown between groups; differences between groups determined by unpaired Welch’s t-test. (C) Representative dot plots of anti-CD3/28-stimulated splenocyte and MLN cultures from Hpb -infected 8–9 week old male WT BALB/c or Jα18−/− mice, with Brefeldin A added to cultures for the last 12 hours. Cells were stained for CD4, Foxp3 and IL4 using Foxp3 staining protocol. Data is representative example of 3 independent experiments for each group.
    Figure Legend Snippet: Helminthic induction and maintenance of TGFβ production requires IL4 production by Foxp3 − CD4 T cells. (A) TGFβ concentration in supernatants of anti-CD3/28-stimulated MLN cultures from Hpb -infected and uninfected 8–9 week old male IL4−/− or WT BALB/c mice, as measured by ELISA. Data show mean±SD from ≥3 independent experiments, with each experiment containing multiple determinations (N indicates the number of independent determinations). p value as indicated on the figure between Hpb -infected vs. uninfected groups; differences between groups determined by unpaired Welch’s t-test. (B) Anti-CD3/28 stimulated MLN T cells from Hpb -infected WT BALB/c mice, as described in Methods were cultured with anti-IL4 blocking (anti-IL4 (+)) isotype control antibodies (Isotype Control), or no antibody added (anti-IL4(–)), as indicated. Supernatants were analyzed for TGFβ content by ELISA. Data show mean±SD from a representative experiment of 5 independent experiments, with each experiment containing multiple determinations; p values as shown between groups; differences between groups determined by unpaired Welch’s t-test. (C) Representative dot plots of anti-CD3/28-stimulated splenocyte and MLN cultures from Hpb -infected 8–9 week old male WT BALB/c or Jα18−/− mice, with Brefeldin A added to cultures for the last 12 hours. Cells were stained for CD4, Foxp3 and IL4 using Foxp3 staining protocol. Data is representative example of 3 independent experiments for each group.

    Techniques Used: Concentration Assay, Infection, Mouse Assay, Enzyme-linked Immunosorbent Assay, Cell Culture, Blocking Assay, Staining

    22) Product Images from "Contact sensitizers trigger human CD1-autoreactive T-cell responses"

    Article Title: Contact sensitizers trigger human CD1-autoreactive T-cell responses

    Journal: European journal of immunology

    doi: 10.1002/eji.201746939

    DNCB activity requires newly synthesized CD1 molecules. (A) Kinetic of THP-1 CD1d cell pulsing with DNCB (6 μM) before stimulation of S33d cells. IFN-γ release was measured by ELISA and data from single experiments representative of 3 independent experiments, are expressed as mean ± SD, n=3. (B) IFN-γ response of S33d cells to THP-1 CD1d cells fixed then pulsed with DNCB (6 μM) or pulsed first with DNCB (6 μM) and then fixed. Data are expressed as mean + SD, n=3, and are from a single experiment representative of 3 independent experiments. (C) IFN-γ response of S33d cells to DNCB (6 μM)- or DMSO vehicle (VEH)-pulsed THP-1 CD1d cells pre-treated with brefeldin A or cycloheximide (Cyclohex). Data are expressed as mean + SD, n=4, and are from a single experiment representative of 2-3 independent experiments. *p 0.05, **p 0.01 ( t -test with Sidak multiple comparisons).
    Figure Legend Snippet: DNCB activity requires newly synthesized CD1 molecules. (A) Kinetic of THP-1 CD1d cell pulsing with DNCB (6 μM) before stimulation of S33d cells. IFN-γ release was measured by ELISA and data from single experiments representative of 3 independent experiments, are expressed as mean ± SD, n=3. (B) IFN-γ response of S33d cells to THP-1 CD1d cells fixed then pulsed with DNCB (6 μM) or pulsed first with DNCB (6 μM) and then fixed. Data are expressed as mean + SD, n=3, and are from a single experiment representative of 3 independent experiments. (C) IFN-γ response of S33d cells to DNCB (6 μM)- or DMSO vehicle (VEH)-pulsed THP-1 CD1d cells pre-treated with brefeldin A or cycloheximide (Cyclohex). Data are expressed as mean + SD, n=4, and are from a single experiment representative of 2-3 independent experiments. *p 0.05, **p 0.01 ( t -test with Sidak multiple comparisons).

    Techniques Used: Activity Assay, Synthesized, Enzyme-linked Immunosorbent Assay

    23) Product Images from "Modulation of Vaccine-Induced HIV-1-Specific Immune Responses by Co-Electroporation of PD-L1 Encoding DNA"

    Article Title: Modulation of Vaccine-Induced HIV-1-Specific Immune Responses by Co-Electroporation of PD-L1 Encoding DNA

    Journal: Vaccines

    doi: 10.3390/vaccines8010027

    Soluble PD-1 and PD-L1 ectodomain co-expression is not inducing an autologous antibody response. 293T cells were transfected with plasmids encoding for Env, Gag, sPD-1, or sPD-L1. Twenty-four hours after transfection, cells were treated with Brefeldin A for 6 h in order to inhibit protein transport. Subsequently, cells were fixed, permeabilized, and incubated with sera from mice immunized with Env- and Gag-DNA either with empty vector (mock) or with corresponding checkpoint ectodomain DNA (sPD-1, sPD-L1). Murine antibodies were detected using an APC-conjugated anti-mouse IgG1 antibody. Shown are the histograms of transfected cells ( A ) as well as the Geometric Mean Fluorescence Intensity of Env- ( B ), Gag- ( C ), sPD-1 ( D ), and sPD-L1 transfected cells ( E ) after incubation with immunized mouse sera and the respective secondary antibody. ( A ) Data are representative of three independent experiments. ( B – E ) Data represent the mean with SEM of one out of three representative experiments with three sera samples from each group.
    Figure Legend Snippet: Soluble PD-1 and PD-L1 ectodomain co-expression is not inducing an autologous antibody response. 293T cells were transfected with plasmids encoding for Env, Gag, sPD-1, or sPD-L1. Twenty-four hours after transfection, cells were treated with Brefeldin A for 6 h in order to inhibit protein transport. Subsequently, cells were fixed, permeabilized, and incubated with sera from mice immunized with Env- and Gag-DNA either with empty vector (mock) or with corresponding checkpoint ectodomain DNA (sPD-1, sPD-L1). Murine antibodies were detected using an APC-conjugated anti-mouse IgG1 antibody. Shown are the histograms of transfected cells ( A ) as well as the Geometric Mean Fluorescence Intensity of Env- ( B ), Gag- ( C ), sPD-1 ( D ), and sPD-L1 transfected cells ( E ) after incubation with immunized mouse sera and the respective secondary antibody. ( A ) Data are representative of three independent experiments. ( B – E ) Data represent the mean with SEM of one out of three representative experiments with three sera samples from each group.

    Techniques Used: Expressing, Transfection, Incubation, Mouse Assay, Plasmid Preparation, Fluorescence

    24) Product Images from "The Immunomodulatory Role of Syncytiotrophoblast Microvesicles"

    Article Title: The Immunomodulatory Role of Syncytiotrophoblast Microvesicles

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0020245

    Monocytes, not B cells, produce the cytokines IL-6, IL-8, TNFα and IL-1β and downregulate HLA-DR in response to pSTBM. A) 10 6 /ml PBMC from non-pregnant donors (n = 3) were stimulated with 50 µg/ml pSTBM, or left untreated, for 20 hours in the presence of Brefeldin A. Intracellular cytokine analysis was performed to detect production of TNFα, IL-6, IL-8 and IL-1β from either B cells (grey bars) or monocytes (black bars). Data shown is the increase in proportion of cells expressing each cytokine above cytokine production in untreated samples, mean (+/− S.D.). B) pSTBM binding to monocytes, but not B cells, caused down-regulation of HLA-DR, shown by median fluorescence intensity of HLA-DR antibody staining (C), (n = 3; mean +/− S.D.).
    Figure Legend Snippet: Monocytes, not B cells, produce the cytokines IL-6, IL-8, TNFα and IL-1β and downregulate HLA-DR in response to pSTBM. A) 10 6 /ml PBMC from non-pregnant donors (n = 3) were stimulated with 50 µg/ml pSTBM, or left untreated, for 20 hours in the presence of Brefeldin A. Intracellular cytokine analysis was performed to detect production of TNFα, IL-6, IL-8 and IL-1β from either B cells (grey bars) or monocytes (black bars). Data shown is the increase in proportion of cells expressing each cytokine above cytokine production in untreated samples, mean (+/− S.D.). B) pSTBM binding to monocytes, but not B cells, caused down-regulation of HLA-DR, shown by median fluorescence intensity of HLA-DR antibody staining (C), (n = 3; mean +/− S.D.).

    Techniques Used: Expressing, Binding Assay, Fluorescence, Staining

    25) Product Images from "Surface receptor Toso controls B cell–mediated regulation of T cell immunity"

    Article Title: Surface receptor Toso controls B cell–mediated regulation of T cell immunity

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI97280

    Toso deficiency results in increased numbers of IL-10–producing B cells. ( A – C ) Purified B cells from WT and Toso –/– (KO) mice were treated with BAFF, LPS, αCD40, or αIgM for 24 hours. For the last 5 hours, cells were stimulated with PMA/ionomycin in the presence of brefeldin A (BFA)/monensin and subsequently analyzed for IL-10 production. ( A ) Representative flow cytometric analysis. ( B and C ) Bar graphs show frequency ( B ) and absolute numbers ( C ) of IL-10–positive B cells. Data are mean ± SEM from 2 cultures derived from different mice. ( D and E ) B cells from mice with straight and conditional Toso knockout, as well as the indicated control mice, were stimulated for 5 hours with LPS and PMA/ionomycin in the presence of BFA/monensin. Frequency ( D ) and number ( E ) of IL-10–positive B cells were determined by intracellular cytokine staining. ( F and G ) WT and Toso –/– (KO) mice were infected i.n. with 50 PFU influenza virus strain A/PR8 (H1N1). At the indicated days p.i., splenocytes were restimulated ex vivo, and the frequency ( F ) and number ( G ) of IL-10–positive CD19 + B cells were quantified by intracellular cytokine staining. ( H ) CD19-Cre +/– mice and Toso f/f /CD19-Cre +/– mice were infected i.n. with 1,000 PFU influenza virus strain A/PR8 (H1N1). Lung cells isolated on day 9 p.i. were restimulated ex vivo, and number and frequency of IL-10–positive CD19 + B cells were quantified by intracellular cytokine staining. Each symbol represents an individual mouse; horizontal lines indicate the mean ± SEM. ( D and E ) n = 3–7; ( F – H ) n = 4–5. * P
    Figure Legend Snippet: Toso deficiency results in increased numbers of IL-10–producing B cells. ( A – C ) Purified B cells from WT and Toso –/– (KO) mice were treated with BAFF, LPS, αCD40, or αIgM for 24 hours. For the last 5 hours, cells were stimulated with PMA/ionomycin in the presence of brefeldin A (BFA)/monensin and subsequently analyzed for IL-10 production. ( A ) Representative flow cytometric analysis. ( B and C ) Bar graphs show frequency ( B ) and absolute numbers ( C ) of IL-10–positive B cells. Data are mean ± SEM from 2 cultures derived from different mice. ( D and E ) B cells from mice with straight and conditional Toso knockout, as well as the indicated control mice, were stimulated for 5 hours with LPS and PMA/ionomycin in the presence of BFA/monensin. Frequency ( D ) and number ( E ) of IL-10–positive B cells were determined by intracellular cytokine staining. ( F and G ) WT and Toso –/– (KO) mice were infected i.n. with 50 PFU influenza virus strain A/PR8 (H1N1). At the indicated days p.i., splenocytes were restimulated ex vivo, and the frequency ( F ) and number ( G ) of IL-10–positive CD19 + B cells were quantified by intracellular cytokine staining. ( H ) CD19-Cre +/– mice and Toso f/f /CD19-Cre +/– mice were infected i.n. with 1,000 PFU influenza virus strain A/PR8 (H1N1). Lung cells isolated on day 9 p.i. were restimulated ex vivo, and number and frequency of IL-10–positive CD19 + B cells were quantified by intracellular cytokine staining. Each symbol represents an individual mouse; horizontal lines indicate the mean ± SEM. ( D and E ) n = 3–7; ( F – H ) n = 4–5. * P

    Techniques Used: Purification, Mouse Assay, Flow Cytometry, Derivative Assay, Knock-Out, Staining, Infection, Ex Vivo, Isolation

    26) Product Images from "Lactobacillus sakei WIKIM30 Ameliorates Atopic Dermatitis-Like Skin Lesions by Inducing Regulatory T Cells and Altering Gut Microbiota Structure in Mice"

    Article Title: Lactobacillus sakei WIKIM30 Ameliorates Atopic Dermatitis-Like Skin Lesions by Inducing Regulatory T Cells and Altering Gut Microbiota Structure in Mice

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.01905

    Inhibitory effect of T helper 2 immune responses in PLN cells of atopic dermatitis (AD) mice treated with WIKIM30.  (A)  PLN cells isolated from each group were treated with PMA/ionomycin/brefeldin A for 4 h and stained for intracellular cytokines [interleukin (IL)-4 and IFN-γ]. The percentages of IFN-γ + CD4 +  and IL-4 + CD4 +  T cells were measured by flow cytometry.  (B,C)  PLN cells were treated with anti-CD3/CD28 monoclonal antibodies for 48 h, and IL-4, IL-5, and IL-13 and IFN-γ levels in the culture supernatant were measured with a cytometric bead array kit. Data represent the mean ± SE of  n  = 5 mice per group from three independent experiments. Student’s  t -test (unpaired); * p
    Figure Legend Snippet: Inhibitory effect of T helper 2 immune responses in PLN cells of atopic dermatitis (AD) mice treated with WIKIM30. (A) PLN cells isolated from each group were treated with PMA/ionomycin/brefeldin A for 4 h and stained for intracellular cytokines [interleukin (IL)-4 and IFN-γ]. The percentages of IFN-γ + CD4 + and IL-4 + CD4 + T cells were measured by flow cytometry. (B,C) PLN cells were treated with anti-CD3/CD28 monoclonal antibodies for 48 h, and IL-4, IL-5, and IL-13 and IFN-γ levels in the culture supernatant were measured with a cytometric bead array kit. Data represent the mean ± SE of n  = 5 mice per group from three independent experiments. Student’s t -test (unpaired); * p

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

    27) Product Images from "Identification of the putative mammalian orthologue of Sec31P, a component of the COPII coat"

    Article Title: Identification of the putative mammalian orthologue of Sec31P, a component of the COPII coat

    Journal: Journal of cell science

    doi:

    p137 and Sec13p remain associated during different experimental treatments. (A–C) Cells transfected with T7-Sec13p plasmid were treated with 10 μg/mL brefeldin A (BFA) for 1 hour at 37°C, then fixed and labeled with mouse anti-p137 antibody (A, p137) and anti-T7 antibody (B, Sec13p). This treatment redistributed the Golgi protein mannosidase II into the ER (not shown). In the presence of brefeldin A, p137 and Sec13p remain co-localized and associated with vesicles (C, merge). (D–F) Cell transfected with T7-Sec13p plasmid were treated with 33 μM nocodazole for 1 h, then fixed and labeled with the same antibodies as described in A. This treatment depolymerized microtubules, as demonstrated by staining with anti-tubulin antibody (not shown); however, p137 (D) and Sec13p (E) remained co-localized (F) but became more widely distributed in the cytoplasm than in control cells.
    Figure Legend Snippet: p137 and Sec13p remain associated during different experimental treatments. (A–C) Cells transfected with T7-Sec13p plasmid were treated with 10 μg/mL brefeldin A (BFA) for 1 hour at 37°C, then fixed and labeled with mouse anti-p137 antibody (A, p137) and anti-T7 antibody (B, Sec13p). This treatment redistributed the Golgi protein mannosidase II into the ER (not shown). In the presence of brefeldin A, p137 and Sec13p remain co-localized and associated with vesicles (C, merge). (D–F) Cell transfected with T7-Sec13p plasmid were treated with 33 μM nocodazole for 1 h, then fixed and labeled with the same antibodies as described in A. This treatment depolymerized microtubules, as demonstrated by staining with anti-tubulin antibody (not shown); however, p137 (D) and Sec13p (E) remained co-localized (F) but became more widely distributed in the cytoplasm than in control cells.

    Techniques Used: Transfection, Plasmid Preparation, Labeling, Staining

    28) Product Images from "Treatment with cyclophosphamide supported by various dendritic cell-based vaccines induces diversification in CD4+ T cell response against MC38 colon carcinoma"

    Article Title: Treatment with cyclophosphamide supported by various dendritic cell-based vaccines induces diversification in CD4+ T cell response against MC38 colon carcinoma

    Journal: International Journal of Oncology

    doi: 10.3892/ijo.2015.3278

    Activity of CD4 + spleen cells. On the 45th day of the experiments, spleens were obtained from CY ± DC-based vaccine-treated mice (N=3–6 per group, two independent experiments). Splenocytes were stimulated for 48 h with Con A (0.5 μg/ml). For the last 6 h, brefeldin A (10 μg/ml) and monensin (2 μM) were added to cell culture. Stimulated spleen cells were fixed, and stained with anti-CD4 antibody and anti-T-bet, anti-GATA3, anti-FoxP3 antibody. As a control, the appropriate isotypes were used. Samples were analyzed by flow cytometry. The percentage of positive cells among CD4 + splenocytes was calculated (mean ± SD). The statistical significance was calculated (for details see Fig. 3 ).
    Figure Legend Snippet: Activity of CD4 + spleen cells. On the 45th day of the experiments, spleens were obtained from CY ± DC-based vaccine-treated mice (N=3–6 per group, two independent experiments). Splenocytes were stimulated for 48 h with Con A (0.5 μg/ml). For the last 6 h, brefeldin A (10 μg/ml) and monensin (2 μM) were added to cell culture. Stimulated spleen cells were fixed, and stained with anti-CD4 antibody and anti-T-bet, anti-GATA3, anti-FoxP3 antibody. As a control, the appropriate isotypes were used. Samples were analyzed by flow cytometry. The percentage of positive cells among CD4 + splenocytes was calculated (mean ± SD). The statistical significance was calculated (for details see Fig. 3 ).

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

    29) Product Images from "The carboxypeptidase angiotensin converting enzyme (ACE) shapes the MHC class I peptide repertoire"

    Article Title: The carboxypeptidase angiotensin converting enzyme (ACE) shapes the MHC class I peptide repertoire

    Journal: Nature Immunology

    doi: 10.1038/ni.2107

    ACE-deficient cells have normal peptide-MHC class I stability but increased peptide supply. ( a ) Peritoneal cells from  Ace +/+  (◆) and  Ace −/−  (□) mice were cultured with brefeldin A. At the indicated time points, cells were stained for either K b  or D b  and analyzed by flow cytometry. Data are plotted as the percentage of similarly cultured cells without brefeldin A. ( b)  Peritoneal cells from  Ace +/+  (◆) and  Ace −/−  (□) mice were treated with mild acid and then cultured in normal medium. Cells were analyzed for K b  and D b  restoration. Data are plotted as percentage of cells not treated with acid. ( a,b ) Data represent means ± s.e.m. of 5 pairs of mice. * P =0.02; ** P ≤0.01.
    Figure Legend Snippet: ACE-deficient cells have normal peptide-MHC class I stability but increased peptide supply. ( a ) Peritoneal cells from Ace +/+ (◆) and Ace −/− (□) mice were cultured with brefeldin A. At the indicated time points, cells were stained for either K b or D b and analyzed by flow cytometry. Data are plotted as the percentage of similarly cultured cells without brefeldin A. ( b) Peritoneal cells from Ace +/+ (◆) and Ace −/− (□) mice were treated with mild acid and then cultured in normal medium. Cells were analyzed for K b and D b restoration. Data are plotted as percentage of cells not treated with acid. ( a,b ) Data represent means ± s.e.m. of 5 pairs of mice. * P =0.02; ** P ≤0.01.

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

    30) Product Images from "AGING INFLUENCES THE RESPONSE OF T CELLS TO STIMULATION BY THE ELLAGITANNIN, OENOTHEIN B"

    Article Title: AGING INFLUENCES THE RESPONSE OF T CELLS TO STIMULATION BY THE ELLAGITANNIN, OENOTHEIN B

    Journal: International immunopharmacology

    doi: 10.1016/j.intimp.2015.04.008

    Oenothein B induces more IFNγ production by bovine CD45RO+ T cells than CD45RO− T cells. PBMCs (10 5 cells/well) isolated from adult cows (4- to- 7 years-old, N=2) were treated with the indicated concentrations of oenothein B or X-VIVO medium alone for 24 hrs, with brefeldin A added for the final 6 hrs. The percentage of CD3+ (A), CD45RO+ (B), or γδ TCR+ (C) cells producing IFNγ was measured by multi-color flow cytometry. The graphs represent pooled data. Each treatment was analyzed in duplicate, and error bars indicate SEM. Significance was determined by Two-way ANOVA with Bonferroni post-test. (D) Representative examples of two-color flow cytometry plots of PBMCs treated with 20 μg/ml oenothein B or medium alone. ***p
    Figure Legend Snippet: Oenothein B induces more IFNγ production by bovine CD45RO+ T cells than CD45RO− T cells. PBMCs (10 5 cells/well) isolated from adult cows (4- to- 7 years-old, N=2) were treated with the indicated concentrations of oenothein B or X-VIVO medium alone for 24 hrs, with brefeldin A added for the final 6 hrs. The percentage of CD3+ (A), CD45RO+ (B), or γδ TCR+ (C) cells producing IFNγ was measured by multi-color flow cytometry. The graphs represent pooled data. Each treatment was analyzed in duplicate, and error bars indicate SEM. Significance was determined by Two-way ANOVA with Bonferroni post-test. (D) Representative examples of two-color flow cytometry plots of PBMCs treated with 20 μg/ml oenothein B or medium alone. ***p

    Techniques Used: Isolation, Flow Cytometry, Cytometry

    Oenothein B induces IFNγ and GM-CSF expression on human adult, but not cord blood, T cells. Human blood mononuclear cells (5×10 4 cells/well) from cord blood (N=3) and adult donors (N=3) were treated with the indicated concentrations of oenothein B or cRPMI medium alone for 24 hrs, and soluble IFNγ (A, left panel) and GM-CSF (B, left panel) levels in supernatant fluids were measured by ELISA. The graph represents pooled data, with each sample plated in triplicate. Human blood mononuclear cells (5×10 4 cells/well) isolated from cord blood (N=1 for IFNγ, N=2 for GM-CSF) and adult (N=1 for IFNγ, N=2 for GM-CSF) donors were also treated with the indicated concentrations of oenothein B or medium alone for 24 hrs, with brefeldin A added for the final 6 hrs. The percent of CD3+ T cells expressing IFNγ (A, right panel) and GM-CSF (B, right panel) was measured by multi-color flow cytometry. Each treatment was analyzed in duplicate, and error bars indicate SEM. Significance between cord blood and adult samples was determined by Two-way ANOVA with Bonferroni post-test. (C) Representative examples of two-color flow cytometry plots of human adult and cord blood mononuclear cells treated with 20 μg/ml oenothein B or medium alone. *p
    Figure Legend Snippet: Oenothein B induces IFNγ and GM-CSF expression on human adult, but not cord blood, T cells. Human blood mononuclear cells (5×10 4 cells/well) from cord blood (N=3) and adult donors (N=3) were treated with the indicated concentrations of oenothein B or cRPMI medium alone for 24 hrs, and soluble IFNγ (A, left panel) and GM-CSF (B, left panel) levels in supernatant fluids were measured by ELISA. The graph represents pooled data, with each sample plated in triplicate. Human blood mononuclear cells (5×10 4 cells/well) isolated from cord blood (N=1 for IFNγ, N=2 for GM-CSF) and adult (N=1 for IFNγ, N=2 for GM-CSF) donors were also treated with the indicated concentrations of oenothein B or medium alone for 24 hrs, with brefeldin A added for the final 6 hrs. The percent of CD3+ T cells expressing IFNγ (A, right panel) and GM-CSF (B, right panel) was measured by multi-color flow cytometry. Each treatment was analyzed in duplicate, and error bars indicate SEM. Significance between cord blood and adult samples was determined by Two-way ANOVA with Bonferroni post-test. (C) Representative examples of two-color flow cytometry plots of human adult and cord blood mononuclear cells treated with 20 μg/ml oenothein B or medium alone. *p

    Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay, Isolation, Flow Cytometry, Cytometry

    31) Product Images from "Stable Isotope Dynamic Labeling of Secretomes (SIDLS) Identifies Authentic Secretory Proteins Released by Cancer and Stromal Cells *"

    Article Title: Stable Isotope Dynamic Labeling of Secretomes (SIDLS) Identifies Authentic Secretory Proteins Released by Cancer and Stromal Cells *

    Journal: Molecular & Cellular Proteomics : MCP

    doi: 10.1074/mcp.TIR117.000516

    Validation of the kinetics of classically secreted proteins by Western blotting. A , The kinetic behavior of three representative classically secreted proteins, MMP1, MMP3 and TGFBI/TGFβig-h3, following pharmacological blockade of secretion in CAMs, were validated using a standard immunoblotting approach. When translation was inhibited with cycloheximide, the accumulation in the medium of MMP1 and TGFβig-h3 was already reduced at 1 h. For these two proteins (and MMP2), secretion was clearly inhibited at 4 h. Similarly, upon perturbation of secretion using brefeldin A (BFA), the appearance of MMP1 was suppressed at 1 h and of all three proteins from 2 h. B , The kinetics of secretion of MMP1, MMP3 and TGFβig-h3 determined by SIDLS clearly match those determined by the orthogonal approach of Western blotting. C , Evidence that SIDLS can discern proteins of the regulated secretory pathway. Western blotting confirmation that secretion of secretogranin-2 (SCG2), an established marker of a minor, endocrine-like secretory phenotype known to be present in stromal myofibroblasts (CAMs), was stimulated by a short (30 min) stimulation with ionomycin. This is consistent with calcium-evoked exocytosis and the response was resistant to cycloheximide (left) and BFA (right), supporting the idea that this represents release from intracellular storage vesicles.
    Figure Legend Snippet: Validation of the kinetics of classically secreted proteins by Western blotting. A , The kinetic behavior of three representative classically secreted proteins, MMP1, MMP3 and TGFBI/TGFβig-h3, following pharmacological blockade of secretion in CAMs, were validated using a standard immunoblotting approach. When translation was inhibited with cycloheximide, the accumulation in the medium of MMP1 and TGFβig-h3 was already reduced at 1 h. For these two proteins (and MMP2), secretion was clearly inhibited at 4 h. Similarly, upon perturbation of secretion using brefeldin A (BFA), the appearance of MMP1 was suppressed at 1 h and of all three proteins from 2 h. B , The kinetics of secretion of MMP1, MMP3 and TGFβig-h3 determined by SIDLS clearly match those determined by the orthogonal approach of Western blotting. C , Evidence that SIDLS can discern proteins of the regulated secretory pathway. Western blotting confirmation that secretion of secretogranin-2 (SCG2), an established marker of a minor, endocrine-like secretory phenotype known to be present in stromal myofibroblasts (CAMs), was stimulated by a short (30 min) stimulation with ionomycin. This is consistent with calcium-evoked exocytosis and the response was resistant to cycloheximide (left) and BFA (right), supporting the idea that this represents release from intracellular storage vesicles.

    Techniques Used: Western Blot, Marker

    32) Product Images from "Interferon-Gamma Impairs Maintenance and Alters Hematopoietic Support of Bone Marrow Mesenchymal Stromal Cells"

    Article Title: Interferon-Gamma Impairs Maintenance and Alters Hematopoietic Support of Bone Marrow Mesenchymal Stromal Cells

    Journal: Stem Cells and Development

    doi: 10.1089/scd.2017.0196

    BM-MSCs are reduced in vivo after IFN-γ exposure. (A) Flow cytometric analysis and (B) quantification of IFN-γ production by BM T cells and NK cells from ARE-Del mice and WT controls. CD4+ and CD8+ T cells were defined as CD3 + CD4 + CD8 − and CD3 + CD4 − CD8 + , respectively. CM = central memory (CD44 + CD62L + ). EM = effector memory (CD44 + CD62 L − ). NK cells were defined as CD3 − CD56 + and NKT cells were CD3 + CD56 + . Cells were incubated for 4 h without any stimulus in the presence of Brefeldin A (mean–SD; n = 2–3). (C) Absolute number of total BM cells and (E) relative numbers of BM stromal cell subsets and BM-MSCs in ARE-Del mice and WT controls (mean–SEM; n = 5). (D) Gating strategy for murine BM MSCs (F) CXCL12 and SCF expression by sorted BM-MSC, normalized to Cyclophilin (mean–SEM; n = 5). (G) Percentage of BM-MSCs in total BM cells of ARE-Del mice and WT controls between 3–30 weeks of age (mean–SEM; n = 3–5). * P
    Figure Legend Snippet: BM-MSCs are reduced in vivo after IFN-γ exposure. (A) Flow cytometric analysis and (B) quantification of IFN-γ production by BM T cells and NK cells from ARE-Del mice and WT controls. CD4+ and CD8+ T cells were defined as CD3 + CD4 + CD8 − and CD3 + CD4 − CD8 + , respectively. CM = central memory (CD44 + CD62L + ). EM = effector memory (CD44 + CD62 L − ). NK cells were defined as CD3 − CD56 + and NKT cells were CD3 + CD56 + . Cells were incubated for 4 h without any stimulus in the presence of Brefeldin A (mean–SD; n = 2–3). (C) Absolute number of total BM cells and (E) relative numbers of BM stromal cell subsets and BM-MSCs in ARE-Del mice and WT controls (mean–SEM; n = 5). (D) Gating strategy for murine BM MSCs (F) CXCL12 and SCF expression by sorted BM-MSC, normalized to Cyclophilin (mean–SEM; n = 5). (G) Percentage of BM-MSCs in total BM cells of ARE-Del mice and WT controls between 3–30 weeks of age (mean–SEM; n = 3–5). * P

    Techniques Used: In Vivo, Flow Cytometry, Mouse Assay, Incubation, Expressing

    33) Product Images from "Neuroinvasive Listeria monocytogenes Infection Triggers IFN-Activation of Microglia and Upregulates Microglial miR-155"

    Article Title: Neuroinvasive Listeria monocytogenes Infection Triggers IFN-Activation of Microglia and Upregulates Microglial miR-155

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.02751

    miR-155 inhibits production of pro-inflammatory cytokines after in vitro stimulation with heat-killed L. monocytogenes . (A) CD11b pos brain cells (2.5 × 10 5 /ml) were collected by immunomagnetic sorting from uninfected B6.WT ( ) and B6.miR-155 −/− ( ) mice and after i.p. infection with 2.1 × 10 5 CFU L. monocytogenes strain EGD, then were incubated overnight with heat-killed L. monocytogenes EGD. Mediator concentrations in cell supernatants were measured by bead array and are presented are the mean ± SD concentration (pg/ml) from 4 mice in each genotype/time point. (B–D) Brain leukocytes from uninfected mice and from mice infected with 2.1–3.2 × 10 5 CFU L. monocytogenes strain EGD were incubated overnight with heat-killed L. monocytogenes EGD, plus Brefeldin A for the last 4–6 h. After immunolabeling of extracellular markers, the cells were fixed and permeabilized then incubated with isotype control or anti-TNF mAb and analyzed by flow cytometry. Microglia were identified as CD45 int CD11b pos cells. TNF pos microglia were identified in each experiment based on gating with isotype control mAb. Dotplots show TNF expression from representative samples including isotype (B) , and 7 d p.i. B6.WT and B6.miR-155 −/− (C) . Percentages of TNF pos microglia from uninfected mice or mice 3 d and 7 d p.i. were normalized to the mean percentage of cytokine pos microglia in B6.WT mice for that experiment and time post-infection. (D) Results from 4 separate experiments are combined and presented as the normalized % TNF pos microglia from uninfected and infected B6.WT ( ) and B6.miR-155 −/− ( ) mice, n = 8–13 mice from each genotype/time point. Symbols represent individual mice with the horizontal bar at the group mean. Significant p -values between genotypes were calculated by 2-tailed Student's t -test are given.
    Figure Legend Snippet: miR-155 inhibits production of pro-inflammatory cytokines after in vitro stimulation with heat-killed L. monocytogenes . (A) CD11b pos brain cells (2.5 × 10 5 /ml) were collected by immunomagnetic sorting from uninfected B6.WT ( ) and B6.miR-155 −/− ( ) mice and after i.p. infection with 2.1 × 10 5 CFU L. monocytogenes strain EGD, then were incubated overnight with heat-killed L. monocytogenes EGD. Mediator concentrations in cell supernatants were measured by bead array and are presented are the mean ± SD concentration (pg/ml) from 4 mice in each genotype/time point. (B–D) Brain leukocytes from uninfected mice and from mice infected with 2.1–3.2 × 10 5 CFU L. monocytogenes strain EGD were incubated overnight with heat-killed L. monocytogenes EGD, plus Brefeldin A for the last 4–6 h. After immunolabeling of extracellular markers, the cells were fixed and permeabilized then incubated with isotype control or anti-TNF mAb and analyzed by flow cytometry. Microglia were identified as CD45 int CD11b pos cells. TNF pos microglia were identified in each experiment based on gating with isotype control mAb. Dotplots show TNF expression from representative samples including isotype (B) , and 7 d p.i. B6.WT and B6.miR-155 −/− (C) . Percentages of TNF pos microglia from uninfected mice or mice 3 d and 7 d p.i. were normalized to the mean percentage of cytokine pos microglia in B6.WT mice for that experiment and time post-infection. (D) Results from 4 separate experiments are combined and presented as the normalized % TNF pos microglia from uninfected and infected B6.WT ( ) and B6.miR-155 −/− ( ) mice, n = 8–13 mice from each genotype/time point. Symbols represent individual mice with the horizontal bar at the group mean. Significant p -values between genotypes were calculated by 2-tailed Student's t -test are given.

    Techniques Used: In Vitro, Mouse Assay, Infection, Incubation, Concentration Assay, Immunolabeling, Flow Cytometry, Cytometry, Expressing

    34) Product Images from "Sample Aging Profoundly Reduces Monocyte Responses in Human Whole Blood Cultures"

    Article Title: Sample Aging Profoundly Reduces Monocyte Responses in Human Whole Blood Cultures

    Journal: Journal of Immunology Research

    doi: 10.1155/2018/8901485

    Cytokine-producing cells in whole blood cultures (average plus SD). The whole blood of 5 donors was stimulated for 3 hours with LPS (a) or PMA/ionomycin (b) in the presence of brefeldin A to block cytokine secretion. The percentage of positive cells for intracellular cytokines was quantified for T-cells (continuous lines) and monocytes (dashed lines). The x -axis indicates the sample age (time span between blood collection and start incubation). The blood was stored at room temperature. T-cells and monocytes remained negative for IFN γ staining after LPS stimulation.
    Figure Legend Snippet: Cytokine-producing cells in whole blood cultures (average plus SD). The whole blood of 5 donors was stimulated for 3 hours with LPS (a) or PMA/ionomycin (b) in the presence of brefeldin A to block cytokine secretion. The percentage of positive cells for intracellular cytokines was quantified for T-cells (continuous lines) and monocytes (dashed lines). The x -axis indicates the sample age (time span between blood collection and start incubation). The blood was stored at room temperature. T-cells and monocytes remained negative for IFN γ staining after LPS stimulation.

    Techniques Used: Blocking Assay, Incubation, Staining

    35) Product Images from "N-glycosylation in the Pre-Membrane Protein Is Essential for the Zika Virus Life Cycle"

    Article Title: N-glycosylation in the Pre-Membrane Protein Is Essential for the Zika Virus Life Cycle

    Journal: Viruses

    doi: 10.3390/v12090925

    The lack of N -glycan on the prME protein induced ER stress. For the immunofluorescence assay, Vero cells were transfected with pcDNA3.1-prME plasmids for 48 h, samples were stained with a mouse anti-CHOP (CCAAT-enhancer-binding protein homologous protein) antibody together with a rabbit anti-ZIKV E antibody followed by secondary antibodies and DAPI. In positive controls, CHOP expression was induced with Brefeldin A (1 µg/mL) or Tunicamycin (1 µg/mL) for 24 h. Representative images of ZIKV E-CHOP double-stained Vero B4 cells. Scale bar = 20 µm.
    Figure Legend Snippet: The lack of N -glycan on the prME protein induced ER stress. For the immunofluorescence assay, Vero cells were transfected with pcDNA3.1-prME plasmids for 48 h, samples were stained with a mouse anti-CHOP (CCAAT-enhancer-binding protein homologous protein) antibody together with a rabbit anti-ZIKV E antibody followed by secondary antibodies and DAPI. In positive controls, CHOP expression was induced with Brefeldin A (1 µg/mL) or Tunicamycin (1 µg/mL) for 24 h. Representative images of ZIKV E-CHOP double-stained Vero B4 cells. Scale bar = 20 µm.

    Techniques Used: Immunofluorescence, Transfection, Staining, Binding Assay, Expressing

    36) Product Images from "Interleukin-27R Signaling Mediates Early Viral Containment and Impacts Innate and Adaptive Immunity after Chronic Lymphocytic Choriomeningitis Virus Infection"

    Article Title: Interleukin-27R Signaling Mediates Early Viral Containment and Impacts Innate and Adaptive Immunity after Chronic Lymphocytic Choriomeningitis Virus Infection

    Journal: Journal of Virology

    doi: 10.1128/JVI.02196-17

    IL-27R signaling regulates NK cell function after LCMV infection. WT and Il27ra −/− mice were infected with 2 × 10 6 PFU of LCMV Cl13 i.v., analyzed at 24 h p.i. (D1), and compared to uninfected mice (U). Spleen NK cells (NK1.1 + CD3 − CD19 − ) were enumerated by flow cytometry (A), along with their expression of granzyme B (GrzB) and IFN-γ (B), Tbet (C), and LAMP-1 (D). Granzyme B, IFN-γ, and LAMP-1 were measured after incubation of splenocytes in the presence of brefeldin A for 5 h. The data are representative of 3 experiments with ≥4 mice per group. The error bars indicate SEM. **, P
    Figure Legend Snippet: IL-27R signaling regulates NK cell function after LCMV infection. WT and Il27ra −/− mice were infected with 2 × 10 6 PFU of LCMV Cl13 i.v., analyzed at 24 h p.i. (D1), and compared to uninfected mice (U). Spleen NK cells (NK1.1 + CD3 − CD19 − ) were enumerated by flow cytometry (A), along with their expression of granzyme B (GrzB) and IFN-γ (B), Tbet (C), and LAMP-1 (D). Granzyme B, IFN-γ, and LAMP-1 were measured after incubation of splenocytes in the presence of brefeldin A for 5 h. The data are representative of 3 experiments with ≥4 mice per group. The error bars indicate SEM. **, P

    Techniques Used: Cell Function Assay, Infection, Mouse Assay, Flow Cytometry, Cytometry, Expressing, Incubation

    37) Product Images from "Analysis of the Trichuris suis excretory/secretory proteins as a function of life cycle stage and their immunomodulatory properties"

    Article Title: Analysis of the Trichuris suis excretory/secretory proteins as a function of life cycle stage and their immunomodulatory properties

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-34174-4

    TsESP-polarized bone marrow-derived cells suppress antigen-specific CD4 + T cell proliferation. BMDC were pretreated for 2 h with either 28-day larvae or adult TsESP, or left untreated (i.e. medium only), then co-cultured with CD4 + T cells in the presence of 1 nM OVA 323–339 peptide. ( A ) Representative profiles of CFSE dilution of gated CD4 + T cells are shown, while proliferation was measured and plotted with five biological replicates for each condition. ( C–E ) Levels of IL-10, IFNγ, and TNFα in supernatants harvested from co-cultures were determined by ELISA. ( F ) CD4 + T cells harvested at 48 h from co-cultures were treated with Brefeldin A for the last 4 h of incubation and stained for intracellular IL-10, IFNγ, and TNFα. Histograms represent the staining of TNFα and IL-10 from the CD4 + IFNγ + cell population. CD11c + BMDC had negligible staining for ICS for any of the cytokines (data not shown).
    Figure Legend Snippet: TsESP-polarized bone marrow-derived cells suppress antigen-specific CD4 + T cell proliferation. BMDC were pretreated for 2 h with either 28-day larvae or adult TsESP, or left untreated (i.e. medium only), then co-cultured with CD4 + T cells in the presence of 1 nM OVA 323–339 peptide. ( A ) Representative profiles of CFSE dilution of gated CD4 + T cells are shown, while proliferation was measured and plotted with five biological replicates for each condition. ( C–E ) Levels of IL-10, IFNγ, and TNFα in supernatants harvested from co-cultures were determined by ELISA. ( F ) CD4 + T cells harvested at 48 h from co-cultures were treated with Brefeldin A for the last 4 h of incubation and stained for intracellular IL-10, IFNγ, and TNFα. Histograms represent the staining of TNFα and IL-10 from the CD4 + IFNγ + cell population. CD11c + BMDC had negligible staining for ICS for any of the cytokines (data not shown).

    Techniques Used: Derivative Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Incubation, Staining

    38) Product Images from "Frequency of CD19+CD24hiCD38hi regulatory B cells is decreased in peripheral blood and synovial fluid of patients with juvenile idiopathic arthritis: a preliminary study"

    Article Title: Frequency of CD19+CD24hiCD38hi regulatory B cells is decreased in peripheral blood and synovial fluid of patients with juvenile idiopathic arthritis: a preliminary study

    Journal: Pediatric Rheumatology Online Journal

    doi: 10.1186/s12969-018-0262-9

    Frequencies of IL-10 producing regulatory B (B10) cells in juvenile idiopathic arthritis (JIA) patients and controls.  a  Representative intracellular IL-10 staining in B cells in peripheral blood (PB) of one JIA patient with/without stimulation of CpG + CD40L for 48 h and phorbol 12-myristate 13-acetate+ionomycin+brefeldin A (PIB) for the last 6 h (right). Few B cells produced IL-10 when cultured with PBS only (left). Isotype controls were used to set up the negative population (lower).  b  Comparison of B10 cells frequencies in PB of different groups of JIA patients, synovial fluid (SF) of JIA patients, and PB of controls (left).  c  Comparison of B10 cells frequencies in active and inactive patients. BFA, brefeldin A; CD40L, CD40 ligand; Iso, isotype; PBMC, peripheral blood mononuclear cells; PBS, phosphate-buffered saline; PIB, phorbol 12-myristate 13-acetate+ionomycin+brefeldin A; poly-JIA, polyarticular juvenile idiopathic arthritis. * p
    Figure Legend Snippet: Frequencies of IL-10 producing regulatory B (B10) cells in juvenile idiopathic arthritis (JIA) patients and controls. a Representative intracellular IL-10 staining in B cells in peripheral blood (PB) of one JIA patient with/without stimulation of CpG + CD40L for 48 h and phorbol 12-myristate 13-acetate+ionomycin+brefeldin A (PIB) for the last 6 h (right). Few B cells produced IL-10 when cultured with PBS only (left). Isotype controls were used to set up the negative population (lower). b Comparison of B10 cells frequencies in PB of different groups of JIA patients, synovial fluid (SF) of JIA patients, and PB of controls (left). c Comparison of B10 cells frequencies in active and inactive patients. BFA, brefeldin A; CD40L, CD40 ligand; Iso, isotype; PBMC, peripheral blood mononuclear cells; PBS, phosphate-buffered saline; PIB, phorbol 12-myristate 13-acetate+ionomycin+brefeldin A; poly-JIA, polyarticular juvenile idiopathic arthritis. * p

    Techniques Used: Staining, Produced, Cell Culture

    39) Product Images from "β-Catenin Signaling Drives Differentiation and Proinflammatory Function of IRF8-Dependent Dendritic Cells"

    Article Title: β-Catenin Signaling Drives Differentiation and Proinflammatory Function of IRF8-Dependent Dendritic Cells

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

    doi: 10.4049/jimmunol.1402453

    CD4 + T cells and NK cells, but not CD8 + T cells, overproduce IFN-γ following Toxoplasma infection in mice with constitutive DC β-catenin signaling. ( A–C ) Intracellular flow cytometric analysis of IFN-γ production by ( A ) CD4 + T cells, ( B ) CD8 + T cells, and ( C ) NK cells from Day 9 T. gondii -infected Ex3 fl/fl and Ex3 DC−/− spleens following 4 hr of PMA and ionomycin stimulation in the presence of Brefeldin-A. Shown are representative contour plots of individual mice and the mean ± standard error of IFN-γ levels from multiple mice. The data are representative of 3 independent experiments (CD4 and CD8) and 1 (NK) experiment. ( D and E ) Quantification of Toxoplasma -specific ( D ) Tgd057 + CD8 + T cells and (E) CD4Ag28m + CD4 + T cells. Means and standard errors of individual mice are shown, and each dot represents a single mouse. ( F and G ) IFN-γ levels expressed by tetramer-positive CD8 + ( F ) and CD4 + ( G ) T cells following 4 hr of culture with PMA, ionomycin, and Brefeldin-A. The data are representative of 3 independent experiments (n=3 mice per group). *, p
    Figure Legend Snippet: CD4 + T cells and NK cells, but not CD8 + T cells, overproduce IFN-γ following Toxoplasma infection in mice with constitutive DC β-catenin signaling. ( A–C ) Intracellular flow cytometric analysis of IFN-γ production by ( A ) CD4 + T cells, ( B ) CD8 + T cells, and ( C ) NK cells from Day 9 T. gondii -infected Ex3 fl/fl and Ex3 DC−/− spleens following 4 hr of PMA and ionomycin stimulation in the presence of Brefeldin-A. Shown are representative contour plots of individual mice and the mean ± standard error of IFN-γ levels from multiple mice. The data are representative of 3 independent experiments (CD4 and CD8) and 1 (NK) experiment. ( D and E ) Quantification of Toxoplasma -specific ( D ) Tgd057 + CD8 + T cells and (E) CD4Ag28m + CD4 + T cells. Means and standard errors of individual mice are shown, and each dot represents a single mouse. ( F and G ) IFN-γ levels expressed by tetramer-positive CD8 + ( F ) and CD4 + ( G ) T cells following 4 hr of culture with PMA, ionomycin, and Brefeldin-A. The data are representative of 3 independent experiments (n=3 mice per group). *, p

    Techniques Used: Infection, Mouse Assay, Flow Cytometry

    40) Product Images from "STAT1 Isoforms Differentially Regulate NK Cell Maturation and Anti-tumor Activity"

    Article Title: STAT1 Isoforms Differentially Regulate NK Cell Maturation and Anti-tumor Activity

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2020.02189

    Stat1 β/β , but not  Stat1 α/α , mice show reduced NK cell-dependent anti-tumor activity,  in vivo  cytotoxicity and impaired production of IFNγ in response to actR stimulation.  (A,B)  1 × 10 6  RMA-Rae1  (A)  or RMA-S  (B)  cells were s.c. injected in both flanks of  WT ,  Stat1 –/– ,  Stat1 α/α , and  Stat1 β/β  mice. Ten days after injection the tumors were isolated and weighed. Tumor weight ( n  = 9–14) and mean values ± SD from two experiments are shown.  (C) WT ,  Stat1 –/– ,  Stat1 α/α , and  Stat1 β/β  mice were i.v. injected with a 1:1 mix of CFSE low  stained  WT  and CFSE high  stained MHC I low  (β 2m –/– ) splenocytes. The ratio of CFSE low  and CFSE high  cells in the spleen of recipient mice was determined 16 h after injection by flow cytometry. Mean percentages ± SD ( n  = 9) from three experiments are given.  (D–F)  Splenocytes from  WT ,  Stat1 –/– ,  Stat1 α/α , and  Stat1 β/β  mice were stimulated with tube-bound anti-NK1.1 antibody  (D,E) , tube-bound anti-NKp46 antibody  (F)  and IL-12 (5 ng/ml) and IL-18 (25 ng/ml)  (G) , and incubated in the presence of brefeldin A. IFNγ production was analyzed by intracellular staining of NK cells  (D,F,G)  or M2 mature NK cells (CD27 – CD11b + )  (E) . Mean percentages ± SEM from six ( n  = 6–18)  (D) , three experiments ( n  = 9)  (E) , one experiment ( n  = 3)  (F) , and two experiments ( n  = 5–6)  (G)  are shown.  (H)  Magnetic beads-purified NK cells were stimulated with IL-12 (5 ng/ml) and IL-18 (25 ng/ml) for 6 h. IFNγ was determined in the culture supernatant by ELISA. Mean IFNγ concentrations ± SEM from one experiment ( n  = 3) is depicted. * p
    Figure Legend Snippet: Stat1 β/β , but not Stat1 α/α , mice show reduced NK cell-dependent anti-tumor activity, in vivo cytotoxicity and impaired production of IFNγ in response to actR stimulation. (A,B) 1 × 10 6 RMA-Rae1 (A) or RMA-S (B) cells were s.c. injected in both flanks of WT , Stat1 –/– , Stat1 α/α , and Stat1 β/β mice. Ten days after injection the tumors were isolated and weighed. Tumor weight ( n = 9–14) and mean values ± SD from two experiments are shown. (C) WT , Stat1 –/– , Stat1 α/α , and Stat1 β/β mice were i.v. injected with a 1:1 mix of CFSE low stained WT and CFSE high stained MHC I low (β 2m –/– ) splenocytes. The ratio of CFSE low and CFSE high cells in the spleen of recipient mice was determined 16 h after injection by flow cytometry. Mean percentages ± SD ( n = 9) from three experiments are given. (D–F) Splenocytes from WT , Stat1 –/– , Stat1 α/α , and Stat1 β/β mice were stimulated with tube-bound anti-NK1.1 antibody (D,E) , tube-bound anti-NKp46 antibody (F) and IL-12 (5 ng/ml) and IL-18 (25 ng/ml) (G) , and incubated in the presence of brefeldin A. IFNγ production was analyzed by intracellular staining of NK cells (D,F,G) or M2 mature NK cells (CD27 – CD11b + ) (E) . Mean percentages ± SEM from six ( n = 6–18) (D) , three experiments ( n = 9) (E) , one experiment ( n = 3) (F) , and two experiments ( n = 5–6) (G) are shown. (H) Magnetic beads-purified NK cells were stimulated with IL-12 (5 ng/ml) and IL-18 (25 ng/ml) for 6 h. IFNγ was determined in the culture supernatant by ELISA. Mean IFNγ concentrations ± SEM from one experiment ( n = 3) is depicted. * p

    Techniques Used: Mouse Assay, Activity Assay, In Vivo, Injection, Isolation, Staining, Flow Cytometry, Incubation, Magnetic Beads, Purification, Enzyme-linked Immunosorbent Assay

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    Flow Cytometry:

    Article Title: Pulmonary Group 2 Innate Lymphoid Cell Phenotype Is Context Specific: Determining the Effect of Strain, Location, and Stimuli
    Article Snippet: .. Flow Cytometry To stain for intracellular cytokines, ~4.5 × 106 total cells were either incubated with PMA (20 ng/ml; Sigma-Aldrich), ionomycin-free acid (1.5 μg/ml) from Streptomyces conglobatus (Merck), and Brefeldin A (5 μg/ml; Sigma-Aldrich) or with Brefeldin A (5 μg/ml) alone or complete media (200 μL) at 37°C for 4 h. Cells were then washed in PBS and stained with LIVE/DEAD Fixable Blue Dead Cell Stain (Thermo Fisher Scientific/Life Technologies) for 20 min at 4°C. .. To stain for extracellular antigens, cells were incubated for 20 min at 4°C with anti-mouse CD16/32 (FC) block (BD Biosciences) and antibodies ( ) in PBS containing 1% bovine serum albumin and 0.01% sodium azide.

    Concentration Assay:

    Article Title: The Multiprotein Exocyst Complex Is Essential for Cell Separation in Schizosaccharomyces pombe
    Article Snippet: .. To eliminate F-actin, yeast cells were treated with latrunculin A (L-12370; Molecular Probes Inc., Eugene, OR) at a concentration of 100 μM for 3.5 h. To block ER-to-Golgi transport, cells were treated with 100 μg/ml Brefeldin A (B-7450; Molecular Probes) for 3 h. Cells treated with DMSO and ethanol, respectively, were used as controls. .. Thiamine was used at a final concentration of 5 μM to repress transcription from the nmt1 promoter ( ).

    Cell Culture:

    Article Title: Toxoplasma Co-infection Prevents Th2 Differentiation and Leads to a Helminth-Specific Th1 Response
    Article Snippet: .. Cells were cultured for 6 days and restimulated with 1 μg/ml PMA and 1 μg/ml Ionomycin (both Sigma-Aldrich, St. Louis, MO, USA) in the presence of 3 μg/ml Brefeldin A (eBioscience, San Diego, CA, USA) followed by intracellular staining. .. Realtime PCR RNA was isolated from intestinal tissue sections previously stored at −80°C via homogenization in RNA lysis buffer.

    Activation Assay:

    Article Title: Inactivation of TRPM7 kinase in mice results in enlarged spleens, reduced T-cell proliferation and diminished store-operated calcium entry
    Article Snippet: .. To determine TNF-α and IL-2 expression, T cells were activated with 0.125 µg/mL PMA and 0.25 µM ionomycin for 6 or 16 hours in the presence of 3 µg/ml Brefeldin A (Thermo Fisher Scientific, eBioscience) during the last 5 and 6 hours for 6 and 16 hr activation conditions, respectively. .. Unstimulated (resting) control cells were treated with Brefeldin A in a similar fashion.

    Incubation:

    Article Title: Pulmonary Group 2 Innate Lymphoid Cell Phenotype Is Context Specific: Determining the Effect of Strain, Location, and Stimuli
    Article Snippet: .. Flow Cytometry To stain for intracellular cytokines, ~4.5 × 106 total cells were either incubated with PMA (20 ng/ml; Sigma-Aldrich), ionomycin-free acid (1.5 μg/ml) from Streptomyces conglobatus (Merck), and Brefeldin A (5 μg/ml; Sigma-Aldrich) or with Brefeldin A (5 μg/ml) alone or complete media (200 μL) at 37°C for 4 h. Cells were then washed in PBS and stained with LIVE/DEAD Fixable Blue Dead Cell Stain (Thermo Fisher Scientific/Life Technologies) for 20 min at 4°C. .. To stain for extracellular antigens, cells were incubated for 20 min at 4°C with anti-mouse CD16/32 (FC) block (BD Biosciences) and antibodies ( ) in PBS containing 1% bovine serum albumin and 0.01% sodium azide.

    other:

    Article Title: Inactivation of TRPM7 kinase in mice results in enlarged spleens, reduced T-cell proliferation and diminished store-operated calcium entry
    Article Snippet: Unstimulated (resting) control cells were treated with Brefeldin A in a similar fashion.

    Blocking Assay:

    Article Title: The Multiprotein Exocyst Complex Is Essential for Cell Separation in Schizosaccharomyces pombe
    Article Snippet: .. To eliminate F-actin, yeast cells were treated with latrunculin A (L-12370; Molecular Probes Inc., Eugene, OR) at a concentration of 100 μM for 3.5 h. To block ER-to-Golgi transport, cells were treated with 100 μg/ml Brefeldin A (B-7450; Molecular Probes) for 3 h. Cells treated with DMSO and ethanol, respectively, were used as controls. .. Thiamine was used at a final concentration of 5 μM to repress transcription from the nmt1 promoter ( ).

    Expressing:

    Article Title: Inactivation of TRPM7 kinase in mice results in enlarged spleens, reduced T-cell proliferation and diminished store-operated calcium entry
    Article Snippet: .. To determine TNF-α and IL-2 expression, T cells were activated with 0.125 µg/mL PMA and 0.25 µM ionomycin for 6 or 16 hours in the presence of 3 µg/ml Brefeldin A (Thermo Fisher Scientific, eBioscience) during the last 5 and 6 hours for 6 and 16 hr activation conditions, respectively. .. Unstimulated (resting) control cells were treated with Brefeldin A in a similar fashion.

    Staining:

    Article Title: Toxoplasma Co-infection Prevents Th2 Differentiation and Leads to a Helminth-Specific Th1 Response
    Article Snippet: .. Cells were cultured for 6 days and restimulated with 1 μg/ml PMA and 1 μg/ml Ionomycin (both Sigma-Aldrich, St. Louis, MO, USA) in the presence of 3 μg/ml Brefeldin A (eBioscience, San Diego, CA, USA) followed by intracellular staining. .. Realtime PCR RNA was isolated from intestinal tissue sections previously stored at −80°C via homogenization in RNA lysis buffer.

    Article Title: Suppression of Autoimmune Retinal Inflammation by an Antiangiogenic Drug
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    Article Title: TCDD, FICZ, and Other High Affinity AhR Ligands Dose-Dependently Determine the Fate of CD4+ T Cell Differentiation
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    Article Title: Pulmonary Group 2 Innate Lymphoid Cell Phenotype Is Context Specific: Determining the Effect of Strain, Location, and Stimuli
    Article Snippet: .. Flow Cytometry To stain for intracellular cytokines, ~4.5 × 106 total cells were either incubated with PMA (20 ng/ml; Sigma-Aldrich), ionomycin-free acid (1.5 μg/ml) from Streptomyces conglobatus (Merck), and Brefeldin A (5 μg/ml; Sigma-Aldrich) or with Brefeldin A (5 μg/ml) alone or complete media (200 μL) at 37°C for 4 h. Cells were then washed in PBS and stained with LIVE/DEAD Fixable Blue Dead Cell Stain (Thermo Fisher Scientific/Life Technologies) for 20 min at 4°C. .. To stain for extracellular antigens, cells were incubated for 20 min at 4°C with anti-mouse CD16/32 (FC) block (BD Biosciences) and antibodies ( ) in PBS containing 1% bovine serum albumin and 0.01% sodium azide.

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    Thermo Fisher brefeldin a
    Expression of IL-2 and TNF-α cytokines in activated KD CD4 + T cells is reduced. ( A , B ) Expression of IL-2 and TNF-α in WT and KD CD4 + splenic T cells, left unstimulated or stimulated for 6 hours with PMA/ionomycin, in the presence of <t>Brefeldin</t> A during the last 5 hrs, determined by flow cytometry. ( C ) Representative flow plots from one independent experiment for data shown in ( B ). ( D , E ) IL-2 and TNF-α expression in resting cells and 16 hrs after PMA/ionomycin stimulation of CD4 + T cells. Brefeldin A was present during the last 6 hrs. For ( A - E ), data were collected from three independent experiments performed on samples from 3 WT and 3 KD mice. Horizontal lines represent arithmetic means. The p value for the differences in the percentage of IL-2 + and TNF-α + cells in WT and KD CD4 + T cells in activated conditions were p = 0.007 ( A ), p = 0.225 ( B ), 0.058 ( D ) and 0.061 ( E ), as determined by Student’s t-test. For resting T cells, the differences between WT and KD in A, B, D and E were not statistically significant.
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    Expression of IL-2 and TNF-α cytokines in activated KD CD4 + T cells is reduced. ( A , B ) Expression of IL-2 and TNF-α in WT and KD CD4 + splenic T cells, left unstimulated or stimulated for 6 hours with PMA/ionomycin, in the presence of Brefeldin A during the last 5 hrs, determined by flow cytometry. ( C ) Representative flow plots from one independent experiment for data shown in ( B ). ( D , E ) IL-2 and TNF-α expression in resting cells and 16 hrs after PMA/ionomycin stimulation of CD4 + T cells. Brefeldin A was present during the last 6 hrs. For ( A - E ), data were collected from three independent experiments performed on samples from 3 WT and 3 KD mice. Horizontal lines represent arithmetic means. The p value for the differences in the percentage of IL-2 + and TNF-α + cells in WT and KD CD4 + T cells in activated conditions were p = 0.007 ( A ), p = 0.225 ( B ), 0.058 ( D ) and 0.061 ( E ), as determined by Student’s t-test. For resting T cells, the differences between WT and KD in A, B, D and E were not statistically significant.

    Journal: Scientific Reports

    Article Title: Inactivation of TRPM7 kinase in mice results in enlarged spleens, reduced T-cell proliferation and diminished store-operated calcium entry

    doi: 10.1038/s41598-018-21004-w

    Figure Lengend Snippet: Expression of IL-2 and TNF-α cytokines in activated KD CD4 + T cells is reduced. ( A , B ) Expression of IL-2 and TNF-α in WT and KD CD4 + splenic T cells, left unstimulated or stimulated for 6 hours with PMA/ionomycin, in the presence of Brefeldin A during the last 5 hrs, determined by flow cytometry. ( C ) Representative flow plots from one independent experiment for data shown in ( B ). ( D , E ) IL-2 and TNF-α expression in resting cells and 16 hrs after PMA/ionomycin stimulation of CD4 + T cells. Brefeldin A was present during the last 6 hrs. For ( A - E ), data were collected from three independent experiments performed on samples from 3 WT and 3 KD mice. Horizontal lines represent arithmetic means. The p value for the differences in the percentage of IL-2 + and TNF-α + cells in WT and KD CD4 + T cells in activated conditions were p = 0.007 ( A ), p = 0.225 ( B ), 0.058 ( D ) and 0.061 ( E ), as determined by Student’s t-test. For resting T cells, the differences between WT and KD in A, B, D and E were not statistically significant.

    Article Snippet: To determine TNF-α and IL-2 expression, T cells were activated with 0.125 µg/mL PMA and 0.25 µM ionomycin for 6 or 16 hours in the presence of 3 µg/ml Brefeldin A (Thermo Fisher Scientific, eBioscience) during the last 5 and 6 hours for 6 and 16 hr activation conditions, respectively.

    Techniques: Expressing, Flow Cytometry, Cytometry, Mouse Assay

    ILC2 marker expression varies with ex vivo re-stimulation. (A) tSNE plot of 4,437 lung Gata3 + cells from both C57BL/6 and BALB/c mice. Representative histograms for extracellular and intracellular markers on Gata3 + cells taken from C57BL/6 and BALB/c mice either left in media or stimulated with brefeldin A (Bref) or PMA, ionomycin and Bref (PIB). Percentage of Gata3 + cells expressing (B) CD25, (C) CD90, (D) CD127, (E) KLRG1, (F) SCA1, (G) ST2, (H) IL-5, and (I) IL-13 and accompanying tSNE plots. Data is the combination of two individual experiments, n = 10 per group. †† P

    Journal: Frontiers in Immunology

    Article Title: Pulmonary Group 2 Innate Lymphoid Cell Phenotype Is Context Specific: Determining the Effect of Strain, Location, and Stimuli

    doi: 10.3389/fimmu.2019.03114

    Figure Lengend Snippet: ILC2 marker expression varies with ex vivo re-stimulation. (A) tSNE plot of 4,437 lung Gata3 + cells from both C57BL/6 and BALB/c mice. Representative histograms for extracellular and intracellular markers on Gata3 + cells taken from C57BL/6 and BALB/c mice either left in media or stimulated with brefeldin A (Bref) or PMA, ionomycin and Bref (PIB). Percentage of Gata3 + cells expressing (B) CD25, (C) CD90, (D) CD127, (E) KLRG1, (F) SCA1, (G) ST2, (H) IL-5, and (I) IL-13 and accompanying tSNE plots. Data is the combination of two individual experiments, n = 10 per group. †† P

    Article Snippet: Flow Cytometry To stain for intracellular cytokines, ~4.5 × 106 total cells were either incubated with PMA (20 ng/ml; Sigma-Aldrich), ionomycin-free acid (1.5 μg/ml) from Streptomyces conglobatus (Merck), and Brefeldin A (5 μg/ml; Sigma-Aldrich) or with Brefeldin A (5 μg/ml) alone or complete media (200 μL) at 37°C for 4 h. Cells were then washed in PBS and stained with LIVE/DEAD Fixable Blue Dead Cell Stain (Thermo Fisher Scientific/Life Technologies) for 20 min at 4°C.

    Techniques: Marker, Expressing, Ex Vivo, Mouse Assay

    Helminthic induction and maintenance of TGFβ production requires IL4 production by Foxp3 − CD4 T cells. (A) TGFβ concentration in supernatants of anti-CD3/28-stimulated MLN cultures from Hpb -infected and uninfected 8–9 week old male IL4−/− or WT BALB/c mice, as measured by ELISA. Data show mean±SD from ≥3 independent experiments, with each experiment containing multiple determinations (N indicates the number of independent determinations). p value as indicated on the figure between Hpb -infected vs. uninfected groups; differences between groups determined by unpaired Welch’s t-test. (B) Anti-CD3/28 stimulated MLN T cells from Hpb -infected WT BALB/c mice, as described in Methods were cultured with anti-IL4 blocking (anti-IL4 (+)) isotype control antibodies (Isotype Control), or no antibody added (anti-IL4(–)), as indicated. Supernatants were analyzed for TGFβ content by ELISA. Data show mean±SD from a representative experiment of 5 independent experiments, with each experiment containing multiple determinations; p values as shown between groups; differences between groups determined by unpaired Welch’s t-test. (C) Representative dot plots of anti-CD3/28-stimulated splenocyte and MLN cultures from Hpb -infected 8–9 week old male WT BALB/c or Jα18−/− mice, with Brefeldin A added to cultures for the last 12 hours. Cells were stained for CD4, Foxp3 and IL4 using Foxp3 staining protocol. Data is representative example of 3 independent experiments for each group.

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

    Article Title: Helminth-Induced Production of TGFβ and Suppression of Graft-Versus-Host Disease Is Dependent on Interleukin-4 Production by Host Cells

    doi: 10.4049/jimmunol.1700638

    Figure Lengend Snippet: Helminthic induction and maintenance of TGFβ production requires IL4 production by Foxp3 − CD4 T cells. (A) TGFβ concentration in supernatants of anti-CD3/28-stimulated MLN cultures from Hpb -infected and uninfected 8–9 week old male IL4−/− or WT BALB/c mice, as measured by ELISA. Data show mean±SD from ≥3 independent experiments, with each experiment containing multiple determinations (N indicates the number of independent determinations). p value as indicated on the figure between Hpb -infected vs. uninfected groups; differences between groups determined by unpaired Welch’s t-test. (B) Anti-CD3/28 stimulated MLN T cells from Hpb -infected WT BALB/c mice, as described in Methods were cultured with anti-IL4 blocking (anti-IL4 (+)) isotype control antibodies (Isotype Control), or no antibody added (anti-IL4(–)), as indicated. Supernatants were analyzed for TGFβ content by ELISA. Data show mean±SD from a representative experiment of 5 independent experiments, with each experiment containing multiple determinations; p values as shown between groups; differences between groups determined by unpaired Welch’s t-test. (C) Representative dot plots of anti-CD3/28-stimulated splenocyte and MLN cultures from Hpb -infected 8–9 week old male WT BALB/c or Jα18−/− mice, with Brefeldin A added to cultures for the last 12 hours. Cells were stained for CD4, Foxp3 and IL4 using Foxp3 staining protocol. Data is representative example of 3 independent experiments for each group.

    Article Snippet: To determine the frequency of IL4 producing cells, splenic or MLN cell cultures were stimulated with anti-CD3/28, and brefeldin A (Thermo Fisher) was added at the last 12 hours to the cultures and at 1:1,000 dilution.

    Techniques: Concentration Assay, Infection, Mouse Assay, Enzyme-linked Immunosorbent Assay, Cell Culture, Blocking Assay, Staining