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    Structured Review

    Thermo Fisher ionomycin
    IL-2/JES6-1 immunocomplex administration promotes intestinal ILC2 expansion and IL-5 expression. WT mice were treated with 3% DSS for 6 days and received normal water thereafter. IL-2/JES6-1 immunocomplexes were delivered every 2 days. IL-5 mRNA expression in total colon tissue was analyzed by quantitative real-time PCR (A) . LPL from each mouse were restimulated by <t>PMA/ionomycin</t> and ILC2 cells were analyzed by flow cytometry. Represent dot plots are shown in (B) and the frequency and total cell numbers are shown in (C) after pre-gating on live, lin- cells. IL-5+ILC2 were analyzed by flow cytometry (D) . Data are representative of two independent experiments with 4–6 mice/group. All data are presented as mean ± SEM; * P
    Ionomycin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 46 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Combined IL-2 Immunocomplex and Anti-IL-5 mAb Treatment Expands Foxp3+ Treg Cells in the Absence of Eosinophilia and Ameliorates Experimental Colitis"

    Article Title: Combined IL-2 Immunocomplex and Anti-IL-5 mAb Treatment Expands Foxp3+ Treg Cells in the Absence of Eosinophilia and Ameliorates Experimental Colitis

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2019.00459

    IL-2/JES6-1 immunocomplex administration promotes intestinal ILC2 expansion and IL-5 expression. WT mice were treated with 3% DSS for 6 days and received normal water thereafter. IL-2/JES6-1 immunocomplexes were delivered every 2 days. IL-5 mRNA expression in total colon tissue was analyzed by quantitative real-time PCR (A) . LPL from each mouse were restimulated by PMA/ionomycin and ILC2 cells were analyzed by flow cytometry. Represent dot plots are shown in (B) and the frequency and total cell numbers are shown in (C) after pre-gating on live, lin- cells. IL-5+ILC2 were analyzed by flow cytometry (D) . Data are representative of two independent experiments with 4–6 mice/group. All data are presented as mean ± SEM; * P
    Figure Legend Snippet: IL-2/JES6-1 immunocomplex administration promotes intestinal ILC2 expansion and IL-5 expression. WT mice were treated with 3% DSS for 6 days and received normal water thereafter. IL-2/JES6-1 immunocomplexes were delivered every 2 days. IL-5 mRNA expression in total colon tissue was analyzed by quantitative real-time PCR (A) . LPL from each mouse were restimulated by PMA/ionomycin and ILC2 cells were analyzed by flow cytometry. Represent dot plots are shown in (B) and the frequency and total cell numbers are shown in (C) after pre-gating on live, lin- cells. IL-5+ILC2 were analyzed by flow cytometry (D) . Data are representative of two independent experiments with 4–6 mice/group. All data are presented as mean ± SEM; * P

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

    2) Product Images from "Tetraspanin CD151 Is a Negative Regulator of FcεRI-Mediated Mast Cell Activation"

    Article Title: Tetraspanin CD151 Is a Negative Regulator of FcεRI-Mediated Mast Cell Activation

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

    doi: 10.4049/jimmunol.1302874

    CD151 deficiency does not affect BMMC degranulation or calcium mobilization. ( A ) β-Hexosaminidase release by stimulated CD151 −/− and WT BMMCs shows no difference in mast cell degranulation ex vivo. Data are represented as the mean ± SEM from three individual assay wells/group over two independent experiments. ( B ) BMMCs were precoated with DNP-IgE overnight and incubated with a dye loading solution (Fluo-4 dye mix, assay buffer, and 5 mM probenecid) and incubated for 60 min prior to 0.5 μg/ml DNP-HSA stimulation. Cells were also stimulated with 0.25 μM ionomycin to confirm the capability of cells to induce calcium influx. Filled columns indicate WT; open columns indicate CD151 −/− .
    Figure Legend Snippet: CD151 deficiency does not affect BMMC degranulation or calcium mobilization. ( A ) β-Hexosaminidase release by stimulated CD151 −/− and WT BMMCs shows no difference in mast cell degranulation ex vivo. Data are represented as the mean ± SEM from three individual assay wells/group over two independent experiments. ( B ) BMMCs were precoated with DNP-IgE overnight and incubated with a dye loading solution (Fluo-4 dye mix, assay buffer, and 5 mM probenecid) and incubated for 60 min prior to 0.5 μg/ml DNP-HSA stimulation. Cells were also stimulated with 0.25 μM ionomycin to confirm the capability of cells to induce calcium influx. Filled columns indicate WT; open columns indicate CD151 −/− .

    Techniques Used: Ex Vivo, Incubation

    3) Product Images from "Immunosuppressive Treg cells acquire the phenotype of effector-T cells in chronic lymphocytic leukemia patients"

    Article Title: Immunosuppressive Treg cells acquire the phenotype of effector-T cells in chronic lymphocytic leukemia patients

    Journal: Journal of Translational Medicine

    doi: 10.1186/s12967-018-1545-0

    Increased Tregs frequency in peripheral blood of CLL patients. a Gating strategy used to identify Tregs as CD4 + CD25 high FoxP3 + . Representative box plots of b Tregs frequency in PBMCs obtained from HV (n = 15) and CLL patients (n = 15); c IL-10-secreting Tregs frequency in PBMCs obtained from HV (n = 15) and CLL patients (n = 15); d Tregs subsets frequency in PBMCs obtained from HV (n = 15) and CLL patients (n = 15), all after in vitro priming with IL-6 and phorbol 12-myristate 13-acetate (P), ionomycin (I) and monensin (M). All results are expressed as median and interquartile range. P value shown is obtained from the comparison between the indicated groups by exact non-parametric Mann–Whitney U test (*P
    Figure Legend Snippet: Increased Tregs frequency in peripheral blood of CLL patients. a Gating strategy used to identify Tregs as CD4 + CD25 high FoxP3 + . Representative box plots of b Tregs frequency in PBMCs obtained from HV (n = 15) and CLL patients (n = 15); c IL-10-secreting Tregs frequency in PBMCs obtained from HV (n = 15) and CLL patients (n = 15); d Tregs subsets frequency in PBMCs obtained from HV (n = 15) and CLL patients (n = 15), all after in vitro priming with IL-6 and phorbol 12-myristate 13-acetate (P), ionomycin (I) and monensin (M). All results are expressed as median and interquartile range. P value shown is obtained from the comparison between the indicated groups by exact non-parametric Mann–Whitney U test (*P

    Techniques Used: In Vitro, MANN-WHITNEY

    4) Product Images from "Cytokine production profile in intestinal mucosa of paediatric inflammatory bowel disease"

    Article Title: Cytokine production profile in intestinal mucosa of paediatric inflammatory bowel disease

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0182313

    Healthy intestinal mucosa contains T lymphocytes prone to secrete proinflammatory mediators only upon a strong in vitro stimuli. T lymphocytes (CD3 positive cells) producing cytokines were analyzed in intestinal biopsies of IBD and control paediatric subjects either at basal condition or after a brief in vitro mitogen stimulation. ( A ) Unstimulated CD3 positive cells infiltration and proportions of CD3 positive cells spontaneously producing TNF-α ( B ), and IFN-γ ( C ) in CD, UC and control biopsies are shown. Frequencies of CD3 positive cells producing TNF-α ( D and E ) and IFN-γ ( F and G ) after stimulation for 5 hours with PMA/Ionomycin are illustrated. Horizontal bars are the median values. The Mann-Whitney U test was applied to evaluate statistical significant differences.
    Figure Legend Snippet: Healthy intestinal mucosa contains T lymphocytes prone to secrete proinflammatory mediators only upon a strong in vitro stimuli. T lymphocytes (CD3 positive cells) producing cytokines were analyzed in intestinal biopsies of IBD and control paediatric subjects either at basal condition or after a brief in vitro mitogen stimulation. ( A ) Unstimulated CD3 positive cells infiltration and proportions of CD3 positive cells spontaneously producing TNF-α ( B ), and IFN-γ ( C ) in CD, UC and control biopsies are shown. Frequencies of CD3 positive cells producing TNF-α ( D and E ) and IFN-γ ( F and G ) after stimulation for 5 hours with PMA/Ionomycin are illustrated. Horizontal bars are the median values. The Mann-Whitney U test was applied to evaluate statistical significant differences.

    Techniques Used: In Vitro, MANN-WHITNEY

    5) Product Images from "The Nrf2 Activator tBHQ Inhibits the Activation of Primary Murine Natural Killer Cells."

    Article Title: The Nrf2 Activator tBHQ Inhibits the Activation of Primary Murine Natural Killer Cells.

    Journal: Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association

    doi: 10.1016/j.fct.2018.08.067

    tBHQ treatment inhibits expression of cell surface molecules CD25 and CD69 in NK cells. Murine splenocytes were isolated and treated with tBHQ (1 μM or 5 μM), VEH (0.01% ethanol), or complete DMEM (BKG) for 30 min, then either unactivated (BKG) or activated by PMA and ionomycin (VEH, 1 μM tBHQ, and 5 μM tBHQ) for 24 h. Cells were labeled with fluorescently conjugated antibodies against CD3ε, NK1.1, CD25, and CD69 for FACS analysis. NK cells were gated on viable cells and identified as CD3 negative and NK1.1 positive prior to analysis of (A) CD25 and (B) CD69 expression. n= 5. * indicates p
    Figure Legend Snippet: tBHQ treatment inhibits expression of cell surface molecules CD25 and CD69 in NK cells. Murine splenocytes were isolated and treated with tBHQ (1 μM or 5 μM), VEH (0.01% ethanol), or complete DMEM (BKG) for 30 min, then either unactivated (BKG) or activated by PMA and ionomycin (VEH, 1 μM tBHQ, and 5 μM tBHQ) for 24 h. Cells were labeled with fluorescently conjugated antibodies against CD3ε, NK1.1, CD25, and CD69 for FACS analysis. NK cells were gated on viable cells and identified as CD3 negative and NK1.1 positive prior to analysis of (A) CD25 and (B) CD69 expression. n= 5. * indicates p

    Techniques Used: Expressing, Isolation, Labeling, FACS

    tBHQ treatment modestly decreases the percentage of NK cells in spleen. Murine splenocytes were isolated and treated with tBHQ (1 μM or 5 μM), VEH (0.01% ethanol), or complete DMEM (BKG) for 30 min, then either unactivated (BKG) or activated by PMA and ionomycin (VEH, 1 μM tBHQ, and 5 μM tBHQ) for 24 h. Cells were then labeled with fluorescently conjugated antibodies against CD3ε and NK1.1 for FACS analysis. NK cells were gated on viable cells and identified as CD3 negative and NK1.1 positive. n= 10. * indicates p
    Figure Legend Snippet: tBHQ treatment modestly decreases the percentage of NK cells in spleen. Murine splenocytes were isolated and treated with tBHQ (1 μM or 5 μM), VEH (0.01% ethanol), or complete DMEM (BKG) for 30 min, then either unactivated (BKG) or activated by PMA and ionomycin (VEH, 1 μM tBHQ, and 5 μM tBHQ) for 24 h. Cells were then labeled with fluorescently conjugated antibodies against CD3ε and NK1.1 for FACS analysis. NK cells were gated on viable cells and identified as CD3 negative and NK1.1 positive. n= 10. * indicates p

    Techniques Used: Isolation, Labeling, FACS

    tBHQ treatment alters NK cell maturation. Murine splenocytes were isolated and treated with tBHQ (1 μM or 5 μM), VEH (0.01% ethanol), or complete DMEM (BKG) for 30 min, then either unactivated (BKG) or activated by PMA and ionomycin (VEH, 1 μM tBHQ, and 5 μM tBHQ) for 24 h. Cells were labeled with fluorescently conjugated antibodies against CD3ε, NK1.1, CD27, and CD11b for FACS analysis. NK cells were gated on viable cells and identified as CD3 negative and NK1.1 positive prior to FACS analysis of maturation markers, CD27 and CD11b. Distinct stages of NK cell maturation occur in the following order: (A) CD27-CD11b-, (B) CD27+CD11b-, (C) CD27+CD11b+, (D) CD27- CD11b+. n= 10. * indicates p
    Figure Legend Snippet: tBHQ treatment alters NK cell maturation. Murine splenocytes were isolated and treated with tBHQ (1 μM or 5 μM), VEH (0.01% ethanol), or complete DMEM (BKG) for 30 min, then either unactivated (BKG) or activated by PMA and ionomycin (VEH, 1 μM tBHQ, and 5 μM tBHQ) for 24 h. Cells were labeled with fluorescently conjugated antibodies against CD3ε, NK1.1, CD27, and CD11b for FACS analysis. NK cells were gated on viable cells and identified as CD3 negative and NK1.1 positive prior to FACS analysis of maturation markers, CD27 and CD11b. Distinct stages of NK cell maturation occur in the following order: (A) CD27-CD11b-, (B) CD27+CD11b-, (C) CD27+CD11b+, (D) CD27- CD11b+. n= 10. * indicates p

    Techniques Used: Isolation, Labeling, FACS

    tBHQ treatment inhibits production of the cytokine IFN Ɣ in NK cells. Murine splenocytes were isolated and treated with tBHQ (1 μM or 5 μM), VEH (0.01% ethanol), or complete DMEM (BKG) for 30 min, then either unactivated (BKG) or activated by PMA and ionomycin (VEH, 1 μM tBHQ, and 5 μM tBHQ) for 24 h. Cells were labeled with fluorescently conjugated antibodies against CD3ε and NK1.1 followed by intracellular labeling of IFN Ɣ with fluorescently conjugated antibodies for FACS analysis. NK cells were gated on viable cells and identified as CD3 negative and NK1.1 positive prior to analysis of IFN Ɣ by (A) percentage and (B) mean fluorescence intensity (MFI). n= 5. * indicates p
    Figure Legend Snippet: tBHQ treatment inhibits production of the cytokine IFN Ɣ in NK cells. Murine splenocytes were isolated and treated with tBHQ (1 μM or 5 μM), VEH (0.01% ethanol), or complete DMEM (BKG) for 30 min, then either unactivated (BKG) or activated by PMA and ionomycin (VEH, 1 μM tBHQ, and 5 μM tBHQ) for 24 h. Cells were labeled with fluorescently conjugated antibodies against CD3ε and NK1.1 followed by intracellular labeling of IFN Ɣ with fluorescently conjugated antibodies for FACS analysis. NK cells were gated on viable cells and identified as CD3 negative and NK1.1 positive prior to analysis of IFN Ɣ by (A) percentage and (B) mean fluorescence intensity (MFI). n= 5. * indicates p

    Techniques Used: Isolation, Labeling, FACS, Fluorescence

    6) Product Images from "Thrombospondin-1 and Angiotensin II Inhibit Soluble Guanylyl Cyclase through an Increase in Intracellular Calcium Concentration"

    Article Title: Thrombospondin-1 and Angiotensin II Inhibit Soluble Guanylyl Cyclase through an Increase in Intracellular Calcium Concentration

    Journal: Biochemistry

    doi: 10.1021/bi201060c

    Representative kinetic plots for immunoprecipitated sGC obtained from MCF-7 cells. Cells were lysed after treatment for 5 min with ionomycin, thapsigargin and 2 mM CaCl 2 , or vehicle control. Reactions were carried out at 37 °C for 10 min (−NO)
    Figure Legend Snippet: Representative kinetic plots for immunoprecipitated sGC obtained from MCF-7 cells. Cells were lysed after treatment for 5 min with ionomycin, thapsigargin and 2 mM CaCl 2 , or vehicle control. Reactions were carried out at 37 °C for 10 min (−NO)

    Techniques Used: Immunoprecipitation

    Increased [Ca 2+ ] i leads to sGC inhibition in Jurkat T cells. A. Manipulation of [Ca 2+ ] i with ionomycin inhibits sGC. Jurkat T cells (1 × 10 6 ) were incubated with the Ca 2+ ionophore ionomycin (1 µg/ml) and the SERCA-inhibitor thapsigargin
    Figure Legend Snippet: Increased [Ca 2+ ] i leads to sGC inhibition in Jurkat T cells. A. Manipulation of [Ca 2+ ] i with ionomycin inhibits sGC. Jurkat T cells (1 × 10 6 ) were incubated with the Ca 2+ ionophore ionomycin (1 µg/ml) and the SERCA-inhibitor thapsigargin

    Techniques Used: Inhibition, Incubation

    7) Product Images from "Phospholipase C, Ca2+, and calmodulin signaling are required for 5-HT2A receptor-mediated transamidation of Rac1 by transglutaminase"

    Article Title: Phospholipase C, Ca2+, and calmodulin signaling are required for 5-HT2A receptor-mediated transamidation of Rac1 by transglutaminase

    Journal: Psychopharmacology

    doi: 10.1007/s00213-010-1984-7

    The Ca 2+ ionophore mimicked the DOI-induced increase in cytosolic Ca 2+ and TGase-catalyzed transamidation of Rac1. a Fura-2-loaded cells were stimulated with either 3 µM DOI or different concentrations of ionomycin. The changes in the fluorescence
    Figure Legend Snippet: The Ca 2+ ionophore mimicked the DOI-induced increase in cytosolic Ca 2+ and TGase-catalyzed transamidation of Rac1. a Fura-2-loaded cells were stimulated with either 3 µM DOI or different concentrations of ionomycin. The changes in the fluorescence

    Techniques Used: Fluorescence

    8) Product Images from "Humanized mouse model supports development, function, and tissue residency of human natural killer cells"

    Article Title: Humanized mouse model supports development, function, and tissue residency of human natural killer cells

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

    doi: 10.1073/pnas.1705301114

    Phenotypic and functional profiling of NK cell subsets in SRG-15 mice and humans by mass cytometry. ( A ) ViSNE single marker 2D scatter plots show mass cytometry-based analysis of CD56 dim CD16 + NK cells in SRG-15 mice ( n = 9; spleen) and humans ( n = 20; blood) following stimulation with K562 tumor cells or PMA plus ionomycin (PMA+I). Thirteen of 33 parameters are shown. Red indicates high expression and dark blue indicates low/absent expression of the respective marker. ( B ) Expression of perforin, CD107a, IFN-γ, MIP-1β, and GM-CSF in NK cell subsets from SRG-15 mice (black) and humans (red). Mean ± SEM are shown. * P
    Figure Legend Snippet: Phenotypic and functional profiling of NK cell subsets in SRG-15 mice and humans by mass cytometry. ( A ) ViSNE single marker 2D scatter plots show mass cytometry-based analysis of CD56 dim CD16 + NK cells in SRG-15 mice ( n = 9; spleen) and humans ( n = 20; blood) following stimulation with K562 tumor cells or PMA plus ionomycin (PMA+I). Thirteen of 33 parameters are shown. Red indicates high expression and dark blue indicates low/absent expression of the respective marker. ( B ) Expression of perforin, CD107a, IFN-γ, MIP-1β, and GM-CSF in NK cell subsets from SRG-15 mice (black) and humans (red). Mean ± SEM are shown. * P

    Techniques Used: Functional Assay, Mouse Assay, Mass Cytometry, Marker, Expressing

    9) Product Images from "Bcl11b, a novel GATA3-interacting protein, suppresses Th1 while limiting Th2 cell differentiation"

    Article Title: Bcl11b, a novel GATA3-interacting protein, suppresses Th1 while limiting Th2 cell differentiation

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20171127

    Bcl11b negatively regulates the production of Th2 cytokines in vitro . (a) Naive CD4 T cells were isolated from Bcl11b fl/fl -CreER T2 mice and cultured under Th1, Th2, or Th17 conditions with or without 4-OHT for 4 d (early). Primed cells were restimulated with PMA and ionomycin for 4 h. Cytokine production was assessed by intracellular staining. (b) Naive CD4 T cells were isolated from Bcl11b fl/fl -CreER T2 mice and cultured under Th2 conditions for 4 d. Then, after resting in IL-2–containing medium for 1 d, the primed cells were further cultured under Th2 conditions (late Th2) with or without 4-OHT for 4 d. Cytokine production was assessed by intracellular staining after restimulation with PMA and ionomycin. Data are representative of three independent experiments. *, P
    Figure Legend Snippet: Bcl11b negatively regulates the production of Th2 cytokines in vitro . (a) Naive CD4 T cells were isolated from Bcl11b fl/fl -CreER T2 mice and cultured under Th1, Th2, or Th17 conditions with or without 4-OHT for 4 d (early). Primed cells were restimulated with PMA and ionomycin for 4 h. Cytokine production was assessed by intracellular staining. (b) Naive CD4 T cells were isolated from Bcl11b fl/fl -CreER T2 mice and cultured under Th2 conditions for 4 d. Then, after resting in IL-2–containing medium for 1 d, the primed cells were further cultured under Th2 conditions (late Th2) with or without 4-OHT for 4 d. Cytokine production was assessed by intracellular staining after restimulation with PMA and ionomycin. Data are representative of three independent experiments. *, P

    Techniques Used: In Vitro, Isolation, Mouse Assay, Cell Culture, Staining

    Bcl11b is important for optimal Th cell proliferation. (a) The expression of Ki-67 in early Th2 cells was measured by intracellular staining. Left peaks are the FMO controls. (b and c) Naive CD4 T cells purified from Bcl11b fl/fl -CreER T2 mice were labeled with CFSE and cultured under Th2 conditions in vitro with or without 4-OHT. After 3 d, cells were restimulated with PMA and ionomycin. IL-4 expression was assessed by intracellular staining. Proliferation index was calculated using FlowJo software. The percentage of IL-4–expressing cells was calculated in cell populations that underwent different cycles of division based on CFSE dilution. (d) RNA-seq analysis was performed with the WT and Bcl11b KO early and late Th2 cells. Cell cycle–related genes regulated by Bcl11b are shown. The total cell cycle gene list (Table S1) is from Qiagen. (e) Early Th2 cells were processed as in b. The expression of p21 and CFSE dilution were assessed. Red, WT Th2 cells; blue, Bcl11b KO Th2 cells. Data are representative of three (a) and two (b, c, and e) independent experiments. *, P
    Figure Legend Snippet: Bcl11b is important for optimal Th cell proliferation. (a) The expression of Ki-67 in early Th2 cells was measured by intracellular staining. Left peaks are the FMO controls. (b and c) Naive CD4 T cells purified from Bcl11b fl/fl -CreER T2 mice were labeled with CFSE and cultured under Th2 conditions in vitro with or without 4-OHT. After 3 d, cells were restimulated with PMA and ionomycin. IL-4 expression was assessed by intracellular staining. Proliferation index was calculated using FlowJo software. The percentage of IL-4–expressing cells was calculated in cell populations that underwent different cycles of division based on CFSE dilution. (d) RNA-seq analysis was performed with the WT and Bcl11b KO early and late Th2 cells. Cell cycle–related genes regulated by Bcl11b are shown. The total cell cycle gene list (Table S1) is from Qiagen. (e) Early Th2 cells were processed as in b. The expression of p21 and CFSE dilution were assessed. Red, WT Th2 cells; blue, Bcl11b KO Th2 cells. Data are representative of three (a) and two (b, c, and e) independent experiments. *, P

    Techniques Used: Expressing, Staining, Purification, Mouse Assay, Labeling, Cell Culture, In Vitro, Software, RNA Sequencing Assay

    10) Product Images from "MFG-E8 Downregulates IL-17 Expression in Sepsis by Modulating STAT3 Activation"

    Article Title: MFG-E8 Downregulates IL-17 Expression in Sepsis by Modulating STAT3 Activation

    Journal: Surgery

    doi: 10.1016/j.surg.2015.08.011

    In vitro stimulation with PMA/ionomycin shows increased IL-17 production in CD4 T-cells isolated from MFG-E8 deficient mice
    Figure Legend Snippet: In vitro stimulation with PMA/ionomycin shows increased IL-17 production in CD4 T-cells isolated from MFG-E8 deficient mice

    Techniques Used: In Vitro, Isolation, Mouse Assay

    In vitro treatment of rmMFG-E8 in splenocytes decreases PMA/ionomycin-induced IL-17 production
    Figure Legend Snippet: In vitro treatment of rmMFG-E8 in splenocytes decreases PMA/ionomycin-induced IL-17 production

    Techniques Used: In Vitro

    11) Product Images from "Malt1 self‐cleavage is critical for regulatory T cell homeostasis and anti‐tumor immunity in mice"

    Article Title: Malt1 self‐cleavage is critical for regulatory T cell homeostasis and anti‐tumor immunity in mice

    Journal: European Journal of Immunology

    doi: 10.1002/eji.201847597

    Malt1 R155A knock‐in mice express a catalytically active form of Malt1 but lack self‐cleavage activity (A) Schematic representation of Malt1 protein and its functional death domain (DD), immunoglobulin‐like domains (Ig), auto processing site and catalytic site. (B) MALT1 protease reporter assays of 293T‐BM cells transiently expressing a mouse wild‐type Malt1 (mp‐Malt1), the self‐cleavage resistant R155A mutant (mp‐Malt1‐SR), the protease‐death C472A mutant (mp‐Malt1‐PD) or empty vector (mock). Malt1 protease‐dependent luciferase activity is shown as fold induction of vector‐transfected cells. The data are shown as mean +SD of four pooled independent experiments, which were each performed in triplicates. ( C ) Cell lysates were immunoblotted with the indicated antibodies to detect MALT1, its N‐ and C‐terminal auto‐cleavage fragments (p16 and p76 respectively) and its proteolytic targets. Arrows indicate the cleavage fragments generated by Malt1 protease activity. (D) Immunoblot analysis of spleen of Malt1 +/+ , Malt1 +/SR , Malt1 SR/SR and Malt1 PD/PD mice. Splenocytes from 4 mice were combined and then incubated with or without PMA and ionomycin for the indicated times. A non‐specific band generated with the Mcpip1 antibody was used a loading control. Data are representative of three independent experiments.
    Figure Legend Snippet: Malt1 R155A knock‐in mice express a catalytically active form of Malt1 but lack self‐cleavage activity (A) Schematic representation of Malt1 protein and its functional death domain (DD), immunoglobulin‐like domains (Ig), auto processing site and catalytic site. (B) MALT1 protease reporter assays of 293T‐BM cells transiently expressing a mouse wild‐type Malt1 (mp‐Malt1), the self‐cleavage resistant R155A mutant (mp‐Malt1‐SR), the protease‐death C472A mutant (mp‐Malt1‐PD) or empty vector (mock). Malt1 protease‐dependent luciferase activity is shown as fold induction of vector‐transfected cells. The data are shown as mean +SD of four pooled independent experiments, which were each performed in triplicates. ( C ) Cell lysates were immunoblotted with the indicated antibodies to detect MALT1, its N‐ and C‐terminal auto‐cleavage fragments (p16 and p76 respectively) and its proteolytic targets. Arrows indicate the cleavage fragments generated by Malt1 protease activity. (D) Immunoblot analysis of spleen of Malt1 +/+ , Malt1 +/SR , Malt1 SR/SR and Malt1 PD/PD mice. Splenocytes from 4 mice were combined and then incubated with or without PMA and ionomycin for the indicated times. A non‐specific band generated with the Mcpip1 antibody was used a loading control. Data are representative of three independent experiments.

    Techniques Used: Knock-In, Mouse Assay, Activity Assay, Functional Assay, Expressing, Mutagenesis, Plasmid Preparation, Luciferase, Transfection, Generated, Incubation

    12) Product Images from "Pulsed infrared radiation excites cultured neonatal spiral and vestibular ganglion neurons by modulating mitochondrial calcium cycling"

    Article Title: Pulsed infrared radiation excites cultured neonatal spiral and vestibular ganglion neurons by modulating mitochondrial calcium cycling

    Journal: Journal of Neurophysiology

    doi: 10.1152/jn.00253.2014

    Ca 520 AM and culture controls. The data are expressed as the 95% confidence interval of the mean ( N = 10 neurons). A : 1 μM ionomycin was used as a positive control of Ca 520 AM. The neurons responded with large transients lasting up to a few
    Figure Legend Snippet: Ca 520 AM and culture controls. The data are expressed as the 95% confidence interval of the mean ( N = 10 neurons). A : 1 μM ionomycin was used as a positive control of Ca 520 AM. The neurons responded with large transients lasting up to a few

    Techniques Used: Positive Control

    13) Product Images from "Glutathione Adducts on Sarcoplasmic/Endoplasmic Reticulum Ca2+ ATPase Cys-674 Regulate Endothelial Cell Calcium Stores and Angiogenic Function as Well as Promote Ischemic Blood Flow Recovery *"

    Article Title: Glutathione Adducts on Sarcoplasmic/Endoplasmic Reticulum Ca2+ ATPase Cys-674 Regulate Endothelial Cell Calcium Stores and Angiogenic Function as Well as Promote Ischemic Blood Flow Recovery *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.554451

    Ca 2+ store maintenance is impaired in SKI endothelial cells. A , release of Ca 2+ stores by ionomycin (1 μ m ) in the absence of extracellular Ca 2+ . ***, p
    Figure Legend Snippet: Ca 2+ store maintenance is impaired in SKI endothelial cells. A , release of Ca 2+ stores by ionomycin (1 μ m ) in the absence of extracellular Ca 2+ . ***, p

    Techniques Used:

    14) Product Images from "Intracellular Localization and Conformational State of Transglutaminase 2: Implications for Cell Death"

    Article Title: Intracellular Localization and Conformational State of Transglutaminase 2: Implications for Cell Death

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0006123

    Transamidating activity of TG2 variants. Graphs showing in situ (a) and in vitro (b) transamidating activities of different TG2 mutants as a function of ionomycin concentration (µM) in the cell culture medium (a) or calcium ion added (mM) to the reaction mixture (b) (N = 3). Results are shown as mean+/−SE *p
    Figure Legend Snippet: Transamidating activity of TG2 variants. Graphs showing in situ (a) and in vitro (b) transamidating activities of different TG2 mutants as a function of ionomycin concentration (µM) in the cell culture medium (a) or calcium ion added (mM) to the reaction mixture (b) (N = 3). Results are shown as mean+/−SE *p

    Techniques Used: Activity Assay, In Situ, In Vitro, Concentration Assay, Cell Culture

    15) Product Images from "The helminth product, ES-62, protects against airway inflammation by resetting the Th cell phenotype"

    Article Title: The helminth product, ES-62, protects against airway inflammation by resetting the Th cell phenotype

    Journal: International Journal for Parasitology

    doi: 10.1016/j.ijpara.2012.12.001

    Neutralising anti-IFNγ antibodies modulate IL-4 responses in ES-62-treated mice undergoing ovalbumin (OVA)-induced airway inflammation. Draining lymph node (DLN) cells from the four indicated treatment groups were cultured with phorbol 12-myristate 13-acetate (PMA) plus ionomycin in the presence of Brefeldin A before staining for intracellular expression of IL-4 prior to FACS analysis: cytokine expression ( y -axis) was plotted against side scatter ( x -axis) and the percentage of DLN cells in the cytokine positive gate shown and annotated to the right of the plot labels (A). Data pooled from two further independent experiments show the numbers (B–F) and Mean Fluorescence Intensity (MFI) (G) of spontaneously- (B, D, G) and PMA plus ionomycin-stimulated (C, E, F) IL-4-producing DLN (B, C), CD4 − DLN (D, E) and CD4 + DLN (F, G) cells. The bar represents the mean value of the group where black ∗ P ⩽ 0.05 represents the difference between the OVA + ES-62 + IgG compared with the OVA + ES-62 + anti-IFNγ group whilst grey ∗ P ⩽ 0.05, ∗∗ P ⩽ 0.01 and ∗∗∗ P ⩽ 0.001 are for PBS compared with indicated treatment group (PBS, n = 6; OVA + IgG, n = 9; OVA + ES-62 + IgG, n = 10; OVA + anti-IFNγ, n = 10 and OVA + ES-62 + anti-IFNγ, n = 9; pooled from two independent experiments).
    Figure Legend Snippet: Neutralising anti-IFNγ antibodies modulate IL-4 responses in ES-62-treated mice undergoing ovalbumin (OVA)-induced airway inflammation. Draining lymph node (DLN) cells from the four indicated treatment groups were cultured with phorbol 12-myristate 13-acetate (PMA) plus ionomycin in the presence of Brefeldin A before staining for intracellular expression of IL-4 prior to FACS analysis: cytokine expression ( y -axis) was plotted against side scatter ( x -axis) and the percentage of DLN cells in the cytokine positive gate shown and annotated to the right of the plot labels (A). Data pooled from two further independent experiments show the numbers (B–F) and Mean Fluorescence Intensity (MFI) (G) of spontaneously- (B, D, G) and PMA plus ionomycin-stimulated (C, E, F) IL-4-producing DLN (B, C), CD4 − DLN (D, E) and CD4 + DLN (F, G) cells. The bar represents the mean value of the group where black ∗ P ⩽ 0.05 represents the difference between the OVA + ES-62 + IgG compared with the OVA + ES-62 + anti-IFNγ group whilst grey ∗ P ⩽ 0.05, ∗∗ P ⩽ 0.01 and ∗∗∗ P ⩽ 0.001 are for PBS compared with indicated treatment group (PBS, n = 6; OVA + IgG, n = 9; OVA + ES-62 + IgG, n = 10; OVA + anti-IFNγ, n = 10 and OVA + ES-62 + anti-IFNγ, n = 9; pooled from two independent experiments).

    Techniques Used: Mouse Assay, Cell Culture, Staining, Expressing, FACS, Fluorescence

    The phosphorylcholine-containing glycoprotein, ES-62, suppresses IL-17 responses in murine ovalbumin (OVA)-induced airway inflammation. Pooled draining lymph node (DLN) cells from the four indicated treatment groups were cultured with medium (A) or phorbol 12-myristate 13-acetate (PMA) plus ionomycin (B) in the presence of Brefeldin A before staining for intracellular expression of IL-17 prior to FACS analysis: cytokine expression ( y -axis) was plotted against side scatter ( x -axis) and the percentage of DLN in the cytokine positive gate shown and annotated to the right of the plots. (C) RORγt expressing unstimulated CD4 + T cells from the DLN of pooled mice from the four treatment groups are shown. The numbers (D) of spontaneously (upper; (PBS , n = 6; OVA + IgG, n = 9; OVA + ES-62 + IgG, n = 10; OVA + anti-IFNγ, n = 10 and OVA + ES-62 + anti-IFNγ, n = 9; pooled from two independent experiments)) and PMA plus ionomycin-stimulated (lower; (PBS, n = 6; OVA + IgG, n = 9; OVA + ES-62 + IgG, n = 9; OVA + anti-IFNγ, n = 10 and OVA + ES-62 + anti-IFNγ, n = 9; pooled from two independent experiments)) IL-17-producing DLN cells and the numbers of IL-17-producing CD4 − DLN cells (E) and proportions of IL-17-producing CD4 + cells (F) from PMA plus ionomycin-stimulated DLN cells from individual mice (PBS, n = 6; OVA + IgG, n = 9; OVA + ES-62 + IgG, n = 9; OVA + anti-IFNγ, n = 10 and OVA + ES-62 + anti-IFNγ, n = 9; pooled from two independent experiments) are presented; the bar represents the mean value of the group and grey ∗ P ⩽ 0.05 shows the difference between the PBS and indicated treatment group. In an independent experiment (G), the levels of IL-17 mRNA (against a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) reference) in the pooled DLN cells from the four indicated treatment groups are shown, normalised to the levels found in the control PBS group. (H) The levels of IL-17 detected by Luminex following stimulation of pooled DLN cells from mice in each group with medium or OVA (500 μg/ml) in a further independent experiment are shown and where ∗∗∗ P ⩽ 0.001 for triplicate samples. In (I), pooled bone marrow-derived dendritic cells (bmDCs) from OVA and OVA + ES-62 mice were pulsed with OVA peptide on day 7 and cocultured with DO11.10 Tg CD4 + CD62L + T cells for 72 h before analysing the levels of IL-17 production in culture supernatants by ELISA. The data are presented as the mean ± S.D. where ∗∗ P ⩽ 0.01, OVA compared with OVA + ES-62 at 30 nM of OVA peptide and ∗∗∗ P ⩽ 0.001, OVA compared with OVA + ES-62 at 300 nm of OVA peptide in one experiment. In a further independent experiment, (J), the levels of IL-4, IFNγ and IL-17 mRNA (against a GAPDH reference) in the lungs from the OVA + ES-62 group (grey bars) are shown, normalised to the levels found in the OVA group (black bars).
    Figure Legend Snippet: The phosphorylcholine-containing glycoprotein, ES-62, suppresses IL-17 responses in murine ovalbumin (OVA)-induced airway inflammation. Pooled draining lymph node (DLN) cells from the four indicated treatment groups were cultured with medium (A) or phorbol 12-myristate 13-acetate (PMA) plus ionomycin (B) in the presence of Brefeldin A before staining for intracellular expression of IL-17 prior to FACS analysis: cytokine expression ( y -axis) was plotted against side scatter ( x -axis) and the percentage of DLN in the cytokine positive gate shown and annotated to the right of the plots. (C) RORγt expressing unstimulated CD4 + T cells from the DLN of pooled mice from the four treatment groups are shown. The numbers (D) of spontaneously (upper; (PBS , n = 6; OVA + IgG, n = 9; OVA + ES-62 + IgG, n = 10; OVA + anti-IFNγ, n = 10 and OVA + ES-62 + anti-IFNγ, n = 9; pooled from two independent experiments)) and PMA plus ionomycin-stimulated (lower; (PBS, n = 6; OVA + IgG, n = 9; OVA + ES-62 + IgG, n = 9; OVA + anti-IFNγ, n = 10 and OVA + ES-62 + anti-IFNγ, n = 9; pooled from two independent experiments)) IL-17-producing DLN cells and the numbers of IL-17-producing CD4 − DLN cells (E) and proportions of IL-17-producing CD4 + cells (F) from PMA plus ionomycin-stimulated DLN cells from individual mice (PBS, n = 6; OVA + IgG, n = 9; OVA + ES-62 + IgG, n = 9; OVA + anti-IFNγ, n = 10 and OVA + ES-62 + anti-IFNγ, n = 9; pooled from two independent experiments) are presented; the bar represents the mean value of the group and grey ∗ P ⩽ 0.05 shows the difference between the PBS and indicated treatment group. In an independent experiment (G), the levels of IL-17 mRNA (against a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) reference) in the pooled DLN cells from the four indicated treatment groups are shown, normalised to the levels found in the control PBS group. (H) The levels of IL-17 detected by Luminex following stimulation of pooled DLN cells from mice in each group with medium or OVA (500 μg/ml) in a further independent experiment are shown and where ∗∗∗ P ⩽ 0.001 for triplicate samples. In (I), pooled bone marrow-derived dendritic cells (bmDCs) from OVA and OVA + ES-62 mice were pulsed with OVA peptide on day 7 and cocultured with DO11.10 Tg CD4 + CD62L + T cells for 72 h before analysing the levels of IL-17 production in culture supernatants by ELISA. The data are presented as the mean ± S.D. where ∗∗ P ⩽ 0.01, OVA compared with OVA + ES-62 at 30 nM of OVA peptide and ∗∗∗ P ⩽ 0.001, OVA compared with OVA + ES-62 at 300 nm of OVA peptide in one experiment. In a further independent experiment, (J), the levels of IL-4, IFNγ and IL-17 mRNA (against a GAPDH reference) in the lungs from the OVA + ES-62 group (grey bars) are shown, normalised to the levels found in the OVA group (black bars).

    Techniques Used: Cell Culture, Staining, Expressing, FACS, Mouse Assay, Luminex, Derivative Assay, Enzyme-linked Immunosorbent Assay

    Exposure to the phosphorylcholine-containing glycoprotein, ES-62, in vivo does not modulate ovalbumin (OVA)-induced elevation of IgG1 and IgG2a but inhibits IgE and suppresses Th2 cells. Serum samples from each mouse were obtained on day 28. Samples were analysed by ELISA for OVA-specific IgE (A) and total IgE (B), at 1/80 and 1/200 dilutions, respectively, and OVA-specific IgG1 (C) and IgG2a (D) over the full titration range where data for each group are presented as the mean ± S.E.M. ( n = 6 mice/group) and ∗∗∗ P ⩽ 0.001, OVA + ES-62 compared with the OVA group. The data are from one experiment representative of two. In E, draining lymph nodes (DLN) cells from each of the four treatment groups were stained for expression of IgE. (F) DLN sections from OVA and OVA + ES-62 groups were stained for B220 + B cells, CD3 + T cells and for the indicated transcription factor (either GATA3, Foxp3 or Tbet) and the transcription factor positive cells were quantified within the T cell regions. Exemplar tissue maps from a single experiment of DLNs from OVA and OVA + ES-62 groups displaying the relevant distribution of such transcription factor positive cells (red) within the T cell paracortical region (white) surrounded by B220 + B cells (blue) and their quantitation of numbers of the relevant transcription factor positive T cells by laser scanning cytometry (LSC) are shown, together with an exemplar relocated image of a portion of the section from the OVA group where Foxp3 is stained in red and CD3, green (F). In an independent experiment, the proportions of Foxp3-expressing unstimulated CD4 + T cells from the DLN of pooled mice from the four treatment groups are shown, with the percentage of cells in the Foxp3 + CD4 + quadrant annotated to the right of the plots (G). Pooled DLN cells from the four treatment groups were cultured with medium (I) or phorbol 12-myristate 13-acetate (PMA) plus ionomycin (IONO) (H, J) in the presence of Brefeldin A before staining for intracellular expression of IL-10 (H) or IFNγ (I, J) prior to FACS analysis: cytokine expression ( y -axis) was plotted against side scatter ( x -axis) and the percentage of DLN cells in the cytokine positive gate is shown and annotated to the right of the plots. (J) The Mean Fluorescence Intensity (MFI) values for IFNγ production for the four treatment groups are PBS: 24,701; ES-62: 21,915; OVA: 23,439 and OVA + ES-62: 27,596. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
    Figure Legend Snippet: Exposure to the phosphorylcholine-containing glycoprotein, ES-62, in vivo does not modulate ovalbumin (OVA)-induced elevation of IgG1 and IgG2a but inhibits IgE and suppresses Th2 cells. Serum samples from each mouse were obtained on day 28. Samples were analysed by ELISA for OVA-specific IgE (A) and total IgE (B), at 1/80 and 1/200 dilutions, respectively, and OVA-specific IgG1 (C) and IgG2a (D) over the full titration range where data for each group are presented as the mean ± S.E.M. ( n = 6 mice/group) and ∗∗∗ P ⩽ 0.001, OVA + ES-62 compared with the OVA group. The data are from one experiment representative of two. In E, draining lymph nodes (DLN) cells from each of the four treatment groups were stained for expression of IgE. (F) DLN sections from OVA and OVA + ES-62 groups were stained for B220 + B cells, CD3 + T cells and for the indicated transcription factor (either GATA3, Foxp3 or Tbet) and the transcription factor positive cells were quantified within the T cell regions. Exemplar tissue maps from a single experiment of DLNs from OVA and OVA + ES-62 groups displaying the relevant distribution of such transcription factor positive cells (red) within the T cell paracortical region (white) surrounded by B220 + B cells (blue) and their quantitation of numbers of the relevant transcription factor positive T cells by laser scanning cytometry (LSC) are shown, together with an exemplar relocated image of a portion of the section from the OVA group where Foxp3 is stained in red and CD3, green (F). In an independent experiment, the proportions of Foxp3-expressing unstimulated CD4 + T cells from the DLN of pooled mice from the four treatment groups are shown, with the percentage of cells in the Foxp3 + CD4 + quadrant annotated to the right of the plots (G). Pooled DLN cells from the four treatment groups were cultured with medium (I) or phorbol 12-myristate 13-acetate (PMA) plus ionomycin (IONO) (H, J) in the presence of Brefeldin A before staining for intracellular expression of IL-10 (H) or IFNγ (I, J) prior to FACS analysis: cytokine expression ( y -axis) was plotted against side scatter ( x -axis) and the percentage of DLN cells in the cytokine positive gate is shown and annotated to the right of the plots. (J) The Mean Fluorescence Intensity (MFI) values for IFNγ production for the four treatment groups are PBS: 24,701; ES-62: 21,915; OVA: 23,439 and OVA + ES-62: 27,596. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

    Techniques Used: In Vivo, Enzyme-linked Immunosorbent Assay, Titration, Mouse Assay, Staining, Expressing, Quantitation Assay, Cytometry, Cell Culture, FACS, Fluorescence

    Exposure to the phosphorylcholine-containing glycoprotein, ES-62, in vivo increases CD4 and CD8 IFNγ responses during murine ovalbumin (OVA)-induced airway inflammation. (A) The proportion of draining lymph node (DLN) cells expressing IFNγ following stimulation with phorbol 12-myristate 13-acetate (PMA) plus ionomycin in the indicated treatment groups by individual mice (PBS, n = 6; OVA + IgG, n = 9; OVA + ES-62 + IgG, n = 10; OVA + anti-IFNγ, n = 10 and OVA + ES-62 + anti-IFNγ, n = 9; pooled from two independent experiments). The bar represents the mean of the group and the OVA + ES-62 + IgG, but not OVA + IgG, group is significantly different from the PBS group (grey ∗∗ P ⩽ 0.01). In an independent experiment, the cytokine expression ( y -axis) by cells from pooled DLN from the indicated groups was analysed and the proportions of IFNγ-producing cells in the lymphocyte gate of DLN cells from mice from the OVA and OVA + ES-62 groups that were CD4 + (B, C) or CD8 + (D, E) following stimulation with medium (B, D) or PMA plus ionomycin (C, E) are shown. The proportions (F) and numbers (G) of PMA plus ionomycin-stimulated CD4 − cells expressing IFNγ in the DLN of individual mice from the indicated groups (PBS, n = 6; OVA + IgG, n = 9; OVA + ES-62 + IgG, n = 10; OVA + anti-IFNγ, n = 10 and OVA + ES-62 + anti-IFNγ, n = 9; pooled from two independent experiments) are shown where the bar represents the mean of the group and the OVA + ES-62 + IgG group demonstrates significantly elevated levels of IFNγ production relative to the PBS control group (where ∗ P ⩽ 0.05). (H) The proportions of PMA plus ionomycin-stimulated CD4 + cells expressing IFNγ in the DLN of individual mice from the indicated groups (PBS, n = 6; OVA + IgG, n = 9; OVA + ES-62 + IgG, n = 10; OVA + anti-IFNγ, n = 10 and OVA + ES-62 + anti-IFNγ, n = 9; pooled from two independent experiments) are shown where the bar represents the mean of the group and no significant differences are detected.
    Figure Legend Snippet: Exposure to the phosphorylcholine-containing glycoprotein, ES-62, in vivo increases CD4 and CD8 IFNγ responses during murine ovalbumin (OVA)-induced airway inflammation. (A) The proportion of draining lymph node (DLN) cells expressing IFNγ following stimulation with phorbol 12-myristate 13-acetate (PMA) plus ionomycin in the indicated treatment groups by individual mice (PBS, n = 6; OVA + IgG, n = 9; OVA + ES-62 + IgG, n = 10; OVA + anti-IFNγ, n = 10 and OVA + ES-62 + anti-IFNγ, n = 9; pooled from two independent experiments). The bar represents the mean of the group and the OVA + ES-62 + IgG, but not OVA + IgG, group is significantly different from the PBS group (grey ∗∗ P ⩽ 0.01). In an independent experiment, the cytokine expression ( y -axis) by cells from pooled DLN from the indicated groups was analysed and the proportions of IFNγ-producing cells in the lymphocyte gate of DLN cells from mice from the OVA and OVA + ES-62 groups that were CD4 + (B, C) or CD8 + (D, E) following stimulation with medium (B, D) or PMA plus ionomycin (C, E) are shown. The proportions (F) and numbers (G) of PMA plus ionomycin-stimulated CD4 − cells expressing IFNγ in the DLN of individual mice from the indicated groups (PBS, n = 6; OVA + IgG, n = 9; OVA + ES-62 + IgG, n = 10; OVA + anti-IFNγ, n = 10 and OVA + ES-62 + anti-IFNγ, n = 9; pooled from two independent experiments) are shown where the bar represents the mean of the group and the OVA + ES-62 + IgG group demonstrates significantly elevated levels of IFNγ production relative to the PBS control group (where ∗ P ⩽ 0.05). (H) The proportions of PMA plus ionomycin-stimulated CD4 + cells expressing IFNγ in the DLN of individual mice from the indicated groups (PBS, n = 6; OVA + IgG, n = 9; OVA + ES-62 + IgG, n = 10; OVA + anti-IFNγ, n = 10 and OVA + ES-62 + anti-IFNγ, n = 9; pooled from two independent experiments) are shown where the bar represents the mean of the group and no significant differences are detected.

    Techniques Used: In Vivo, Expressing, Mouse Assay

    16) Product Images from "Herpes simplex viruses activate phospholipid scramblase to redistribute phosphatidylserines and Akt to the outer leaflet of the plasma membrane and promote viral entry"

    Article Title: Herpes simplex viruses activate phospholipid scramblase to redistribute phosphatidylserines and Akt to the outer leaflet of the plasma membrane and promote viral entry

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1006766

    Phospholipid scramblase blockade prevents HSV-induced Akt phosphorylation, Ca 2+ release and viral entry. (A) CaSki cells were transfected with siControl or siPLSCR1 and 72 h post-transfection were infected with purified HSV-2(G) (10 pfu/cell). Western blots of cell lysates harvested at the indicated times post-infection were probed with anti- pS 473 -Akt and then stripped and probed with anti-total Akt and anti-PLSCR1 antibodies. Representative blots from 2 independent experiments are shown. Images were scanned and pAkt as a percentage of total Akt was calculated (mean+ SEM). (B) CaSki cells were transfected with siRNA as in (A), loaded with Fura-2 and then infected with purified HSV-2(G) (5 pfu/cell), HSV-1(KOS) or mock-infected and the kinetics of calcium response monitored over 60 minutes. (C) The mean calcium released over 1 h was calculated from 4 wells in 3 independent experiments, each containing 5 x10 4 cells. As additional controls, siRNA transfected cells were treated with ionomycin. The asterisks indicate significant differences in Ca 2+ concentration relative to mock-infected controls (*, p
    Figure Legend Snippet: Phospholipid scramblase blockade prevents HSV-induced Akt phosphorylation, Ca 2+ release and viral entry. (A) CaSki cells were transfected with siControl or siPLSCR1 and 72 h post-transfection were infected with purified HSV-2(G) (10 pfu/cell). Western blots of cell lysates harvested at the indicated times post-infection were probed with anti- pS 473 -Akt and then stripped and probed with anti-total Akt and anti-PLSCR1 antibodies. Representative blots from 2 independent experiments are shown. Images were scanned and pAkt as a percentage of total Akt was calculated (mean+ SEM). (B) CaSki cells were transfected with siRNA as in (A), loaded with Fura-2 and then infected with purified HSV-2(G) (5 pfu/cell), HSV-1(KOS) or mock-infected and the kinetics of calcium response monitored over 60 minutes. (C) The mean calcium released over 1 h was calculated from 4 wells in 3 independent experiments, each containing 5 x10 4 cells. As additional controls, siRNA transfected cells were treated with ionomycin. The asterisks indicate significant differences in Ca 2+ concentration relative to mock-infected controls (*, p

    Techniques Used: Transfection, Infection, Purification, Western Blot, Concentration Assay

    Ionomycin activates phospholipid scramblase leading to externalization of phosphatidylserines and Akt. (A). CaSki cells were treated with ionomycin (1μM), HSV-2(G) (MOI 5 pfu/cell) or DMSO (0.1%) for 15 minutes and then fixed and stained with antibodies to PLSCR1(polyclonal rabbit and Alexa 555, red), PtdS (monoclonal antibody and Alexa555, red), or Akt (polyclonal rabbit and secondary Alexa488, green) as indicated. Nuclei were stained blue with DAPI. Images are representative of results obtained from 2–3 independent experiments; bar = 10μm. Five fields were scanned and mean fluorescence intensity (MFI) per cell calculated using ImageJ software (NIH); results are mean +SEM and the asterisks indicate significant differences by ANOVA compared to DMSO control treated cells. (B). CaSki cells were treated as in A for 15 minutes and then cell lysates were harvested, incubated with a goat anti-PLSCR1 antibody and immune complexes precipitated with protein G-agarose and analyzed by Western blotting with a mouse anti-phosphotyrosine mAb (PY20) or mouse anti-PLSCR1 Ab. The blot is representative of results obtained in 2 independent experiments. (C). CaSki cells were harvested 5 or 15 minutes after exposure to DMSO, 1μM ionomycin (Iono) or HSV-2(G) (5 pfu/cell). Cell surface proteins were biotinylated and precipitated with streptavidin magnetic beads and the pellet analyzed by immunoblotting with rabbit anti-pAktThr 308 or mouse anti-pAktS 473 Abs. In parallel, cellular lysates were analyzed by immunoblotting for total cellular Akt (rabbit polyclonal Ab). Blots representative of 2 independent experiments are shown. (D). The blots were scanned and fold increase in pAktThr 308 and anti-pAktS 473 relative to DMSO treated cells is depicted (mean+ SEM). (E). HaCAT cells were treated with 1μM ionomycin or DMSO (0.1%) for 10 minutes and then incubated with soluble gL for 30 minutes at 37°C, transferred to ice, and immunoprecipitated (IP) with goat anti-PLSCR1 (left) or with mouse-anti-gL (right). Equivalent volumes of whole cell lysate, supernatant, or pellet (L, S, P) were analyzed by preparing Western blots (WB) and probing with mouse anti-gL, rabbit anti-PLSCR1 or rabbit anti-FIC-1 as a control. The blots are representative of results obtained in 2 independent experiments.
    Figure Legend Snippet: Ionomycin activates phospholipid scramblase leading to externalization of phosphatidylserines and Akt. (A). CaSki cells were treated with ionomycin (1μM), HSV-2(G) (MOI 5 pfu/cell) or DMSO (0.1%) for 15 minutes and then fixed and stained with antibodies to PLSCR1(polyclonal rabbit and Alexa 555, red), PtdS (monoclonal antibody and Alexa555, red), or Akt (polyclonal rabbit and secondary Alexa488, green) as indicated. Nuclei were stained blue with DAPI. Images are representative of results obtained from 2–3 independent experiments; bar = 10μm. Five fields were scanned and mean fluorescence intensity (MFI) per cell calculated using ImageJ software (NIH); results are mean +SEM and the asterisks indicate significant differences by ANOVA compared to DMSO control treated cells. (B). CaSki cells were treated as in A for 15 minutes and then cell lysates were harvested, incubated with a goat anti-PLSCR1 antibody and immune complexes precipitated with protein G-agarose and analyzed by Western blotting with a mouse anti-phosphotyrosine mAb (PY20) or mouse anti-PLSCR1 Ab. The blot is representative of results obtained in 2 independent experiments. (C). CaSki cells were harvested 5 or 15 minutes after exposure to DMSO, 1μM ionomycin (Iono) or HSV-2(G) (5 pfu/cell). Cell surface proteins were biotinylated and precipitated with streptavidin magnetic beads and the pellet analyzed by immunoblotting with rabbit anti-pAktThr 308 or mouse anti-pAktS 473 Abs. In parallel, cellular lysates were analyzed by immunoblotting for total cellular Akt (rabbit polyclonal Ab). Blots representative of 2 independent experiments are shown. (D). The blots were scanned and fold increase in pAktThr 308 and anti-pAktS 473 relative to DMSO treated cells is depicted (mean+ SEM). (E). HaCAT cells were treated with 1μM ionomycin or DMSO (0.1%) for 10 minutes and then incubated with soluble gL for 30 minutes at 37°C, transferred to ice, and immunoprecipitated (IP) with goat anti-PLSCR1 (left) or with mouse-anti-gL (right). Equivalent volumes of whole cell lysate, supernatant, or pellet (L, S, P) were analyzed by preparing Western blots (WB) and probing with mouse anti-gL, rabbit anti-PLSCR1 or rabbit anti-FIC-1 as a control. The blots are representative of results obtained in 2 independent experiments.

    Techniques Used: Staining, Fluorescence, Software, Incubation, Western Blot, Magnetic Beads, Immunoprecipitation

    17) Product Images from "Transgenic Eimeria magna Pérard, 1925 Displays Similar Parasitological Properties to the Wild-type Strain and Induces an Exogenous Protein-Specific Immune Response in Rabbits (Oryctolagus cuniculus L.)"

    Article Title: Transgenic Eimeria magna Pérard, 1925 Displays Similar Parasitological Properties to the Wild-type Strain and Induces an Exogenous Protein-Specific Immune Response in Rabbits (Oryctolagus cuniculus L.)

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2017.00002

    Transcriptional levels of Th1 cytokines of splenocytes and mesenteric lymph nodes (MLN) of rabbits immunized with EmagWT and EmagER after in vitro stimulation . Three groups of rabbits were inoculated with either 200 oocysts of EmagWT, Em ag ER , or 200 µl PBS, respectively. Splenocytes and MLN lymphocytes were isolated and stimulated with 20 µg/ml of either EmagWT-soluble antigen, recombinant enhanced yellow fluorescent protein, recombinant red fluorescent protein, or positive stimulus (P, containing 40.5 µM PMA and 670 µM ionomycin). Quantitative real-time PCR was performed using GAPDH as an internal control. Relative gene expression was calculated by the 2 −ΔΔCq (Livak) method. All data were presented as mean ± SEM values. Statistical analysis was performed by one-way ANOVA, LSD.
    Figure Legend Snippet: Transcriptional levels of Th1 cytokines of splenocytes and mesenteric lymph nodes (MLN) of rabbits immunized with EmagWT and EmagER after in vitro stimulation . Three groups of rabbits were inoculated with either 200 oocysts of EmagWT, Em ag ER , or 200 µl PBS, respectively. Splenocytes and MLN lymphocytes were isolated and stimulated with 20 µg/ml of either EmagWT-soluble antigen, recombinant enhanced yellow fluorescent protein, recombinant red fluorescent protein, or positive stimulus (P, containing 40.5 µM PMA and 670 µM ionomycin). Quantitative real-time PCR was performed using GAPDH as an internal control. Relative gene expression was calculated by the 2 −ΔΔCq (Livak) method. All data were presented as mean ± SEM values. Statistical analysis was performed by one-way ANOVA, LSD.

    Techniques Used: In Vitro, Isolation, Recombinant, Real-time Polymerase Chain Reaction, Expressing

    18) Product Images from "Anaphylactic shock depends on endothelial Gq/G11"

    Article Title: Anaphylactic shock depends on endothelial Gq/G11

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20082150

    The role of G q /G 11 and G 12 /G 13 in the regulation of NO production and MLC phosphorylation in pulmonary ECs. (A) Lysates of pulmonary ECs prepared from WT, Gnaq flox/flox ; Gna11 −/− (q/11-KO), or Gna12 −/− ; Gna13 flox/flox (12/13-KO) mice were infected with Cre-transducing adenovirus and were analyzed by Western blotting with antibodies directed against Gα q /Gα 11 , Gα 13 , or α-tubulin. Arrowheads indicate the position of the 43-kD marker protein. The presented data are representative of at least five experiments performed with samples from different animals. (B) WT Gα q /Gα 11 -deficient (q/11-KO) and Gα 12 /Gα 13 -deficient (12/13-KO) ECs were incubated without and with 1 U/ml thrombin (thromb.), 100 nM PAF, or 100 nM ionomycin (ionom.), and NO bioavailability was assessed in a transfer bioassay by determining cGMP production in detector RFL6 fibroblasts by radioimmunoassay. Shown are the results of three separate experiments (mean values ± SEM). (C–E) WT, Gα q /Gα 11 - (q/11-KO), and Gα 12 /Gα 13 -deficient (12/13-KO) ECs were incubated in the absence or presence of 1 U/ml thrombin for 1, 3, or 10 min, and the amount of phosphorylated MLC (pMLC) was determined using a phosphorylation site-specific antibody (see Materials and methods). Where indicated (Ad-Gα q +), cells had been transfected with Gα q using an adenoviral transfection system. Shown are representative Western blots of cell lysates using the indicated antibodies (C and D) and the results of the densitometric evaluation of three independently performed experiments (E). Shown are mean values ± SEM. Arrowheads indicate the position of the 25- or 43-kD (D, bottom) marker proteins. (F) Effect of 1 U/ml thrombin on RhoA activity in WT, Gα q /Gα 11 -deficient (q/11-KO), and Gα 12 /Gα 13 -deficient lung ECs (12/13-KO). Data are from three independently performed experiments (mean values ± SD).
    Figure Legend Snippet: The role of G q /G 11 and G 12 /G 13 in the regulation of NO production and MLC phosphorylation in pulmonary ECs. (A) Lysates of pulmonary ECs prepared from WT, Gnaq flox/flox ; Gna11 −/− (q/11-KO), or Gna12 −/− ; Gna13 flox/flox (12/13-KO) mice were infected with Cre-transducing adenovirus and were analyzed by Western blotting with antibodies directed against Gα q /Gα 11 , Gα 13 , or α-tubulin. Arrowheads indicate the position of the 43-kD marker protein. The presented data are representative of at least five experiments performed with samples from different animals. (B) WT Gα q /Gα 11 -deficient (q/11-KO) and Gα 12 /Gα 13 -deficient (12/13-KO) ECs were incubated without and with 1 U/ml thrombin (thromb.), 100 nM PAF, or 100 nM ionomycin (ionom.), and NO bioavailability was assessed in a transfer bioassay by determining cGMP production in detector RFL6 fibroblasts by radioimmunoassay. Shown are the results of three separate experiments (mean values ± SEM). (C–E) WT, Gα q /Gα 11 - (q/11-KO), and Gα 12 /Gα 13 -deficient (12/13-KO) ECs were incubated in the absence or presence of 1 U/ml thrombin for 1, 3, or 10 min, and the amount of phosphorylated MLC (pMLC) was determined using a phosphorylation site-specific antibody (see Materials and methods). Where indicated (Ad-Gα q +), cells had been transfected with Gα q using an adenoviral transfection system. Shown are representative Western blots of cell lysates using the indicated antibodies (C and D) and the results of the densitometric evaluation of three independently performed experiments (E). Shown are mean values ± SEM. Arrowheads indicate the position of the 25- or 43-kD (D, bottom) marker proteins. (F) Effect of 1 U/ml thrombin on RhoA activity in WT, Gα q /Gα 11 -deficient (q/11-KO), and Gα 12 /Gα 13 -deficient lung ECs (12/13-KO). Data are from three independently performed experiments (mean values ± SD).

    Techniques Used: Mouse Assay, Infection, Western Blot, Marker, Incubation, RIA Assay, Transfection, Activity Assay

    19) Product Images from "Variation in human cancer cell external phosphatidylserine is regulated by flippase activity and intracellular calcium"

    Article Title: Variation in human cancer cell external phosphatidylserine is regulated by flippase activity and intracellular calcium

    Journal: Oncotarget

    doi:

    Modulation of intracellular calcium by ionomycin or BAPTA-AM affects surface PS in human cancer cells A . Cells were treated with DMSO, ionomycin or BAPTA-AM, incubated with the calcium binding dye Fluo-4 Direct and fluorescence was measured using a microplate reader. Bars show relative changes in Fluo-4 Direct fluorescence signal for each cell line normalized to DMSO controls. B . Low and high surface PS cells were treated with DMSO or ionomycin and surface PS levels were assayed by staining with annexin V FITC, followed by flow cytometry. The axis indicates shift in the geometrical mean of annexin V FITC fluorescence in ionomycin treated vs untreated cells (U87ΔEGFR-Luc □, H1299 Δ, MDA-MB-231 ○, MDA-MB-231-Luc-D3H2LN ◆, Gli36 ▲, U373 ●, Mean—). (C) Low and and high surface PS cells were treated with DMSO or BAPTA-AM, followed by incubation with annexin V FITC and analyzed by flow cytometry. The axis indicates shift in the geometrical mean of annexin V FITC fluorescence in BAPTA-AM treated vs untreated cells (U87ΔEGFR-Luc □, H1299 Δ, MDA-MB-231 ○, MDA-MB-231-Luc-D3H2LN ◆, Gli36 ▲, U373 ●, Mean—).
    Figure Legend Snippet: Modulation of intracellular calcium by ionomycin or BAPTA-AM affects surface PS in human cancer cells A . Cells were treated with DMSO, ionomycin or BAPTA-AM, incubated with the calcium binding dye Fluo-4 Direct and fluorescence was measured using a microplate reader. Bars show relative changes in Fluo-4 Direct fluorescence signal for each cell line normalized to DMSO controls. B . Low and high surface PS cells were treated with DMSO or ionomycin and surface PS levels were assayed by staining with annexin V FITC, followed by flow cytometry. The axis indicates shift in the geometrical mean of annexin V FITC fluorescence in ionomycin treated vs untreated cells (U87ΔEGFR-Luc □, H1299 Δ, MDA-MB-231 ○, MDA-MB-231-Luc-D3H2LN ◆, Gli36 ▲, U373 ●, Mean—). (C) Low and and high surface PS cells were treated with DMSO or BAPTA-AM, followed by incubation with annexin V FITC and analyzed by flow cytometry. The axis indicates shift in the geometrical mean of annexin V FITC fluorescence in BAPTA-AM treated vs untreated cells (U87ΔEGFR-Luc □, H1299 Δ, MDA-MB-231 ○, MDA-MB-231-Luc-D3H2LN ◆, Gli36 ▲, U373 ●, Mean—).

    Techniques Used: Incubation, Binding Assay, Fluorescence, Staining, Flow Cytometry, Cytometry, Multiple Displacement Amplification

    20) Product Images from "Skeletal Muscle Expression of the Adhesion-GPCR CD97: CD97 Deletion Induces an Abnormal Structure of the Sarcoplasmatic Reticulum but Does Not Impair Skeletal Muscle Function"

    Article Title: Skeletal Muscle Expression of the Adhesion-GPCR CD97: CD97 Deletion Induces an Abnormal Structure of the Sarcoplasmatic Reticulum but Does Not Impair Skeletal Muscle Function

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0100513

    Calcium release from myofibers and muscle force generation and fatigability are normal in CD97Ko mice. A–D Intracellular Ca 2+ release from the SR into cytoplasm of single muscle flexor digitorum brevis (FDB) myofibers. Mean traces of the analyzed FDB fibers from adult WT and CD97Ko represent [Ca 2+ ]i responses measured by Fura-2AM video microscopy. Variations in [Ca 2+ ]i over time are represented by the ratio fluorescence intensities (FI) at 340 and 380 nm excitation wavelengths after dynamic background subtraction. Fibers were exposed to calcium release activators after 60 s. From 120 s till 300 s, medium without a substance was applied again. 5 mice per strain and from each mouse two to three fibers were analyzed (mean ± SEM, Mann-Whitney test). A Basal 340/380 fluorescence intensities (FI) ratios were unchanged in both WT and CD97ko fibers. B, C 100 mM KCl (B) and 30 mM caffeine (C) increased [Ca 2+ ]i comparably in WT and CD97Ko fibers. D Application of 5 µM ionomycin as a positive control induced a fast total store [Ca 2+ ]i release from the SR. E, F Functional analysis of skeletal muscles of CD97Ko and WT mice at the age of 2 and 4 months. Force-frequency relationship (E) and muscle fatigability, determined as percentage of decline in force over 15 s (F), were measured in soleus muscles (n = 8 mice/strain; mean ± SEM, t-test).
    Figure Legend Snippet: Calcium release from myofibers and muscle force generation and fatigability are normal in CD97Ko mice. A–D Intracellular Ca 2+ release from the SR into cytoplasm of single muscle flexor digitorum brevis (FDB) myofibers. Mean traces of the analyzed FDB fibers from adult WT and CD97Ko represent [Ca 2+ ]i responses measured by Fura-2AM video microscopy. Variations in [Ca 2+ ]i over time are represented by the ratio fluorescence intensities (FI) at 340 and 380 nm excitation wavelengths after dynamic background subtraction. Fibers were exposed to calcium release activators after 60 s. From 120 s till 300 s, medium without a substance was applied again. 5 mice per strain and from each mouse two to three fibers were analyzed (mean ± SEM, Mann-Whitney test). A Basal 340/380 fluorescence intensities (FI) ratios were unchanged in both WT and CD97ko fibers. B, C 100 mM KCl (B) and 30 mM caffeine (C) increased [Ca 2+ ]i comparably in WT and CD97Ko fibers. D Application of 5 µM ionomycin as a positive control induced a fast total store [Ca 2+ ]i release from the SR. E, F Functional analysis of skeletal muscles of CD97Ko and WT mice at the age of 2 and 4 months. Force-frequency relationship (E) and muscle fatigability, determined as percentage of decline in force over 15 s (F), were measured in soleus muscles (n = 8 mice/strain; mean ± SEM, t-test).

    Techniques Used: Mouse Assay, Microscopy, Fluorescence, MANN-WHITNEY, Positive Control, Functional Assay

    21) Product Images from "The Immune Cell Composition in Barrett's Metaplastic Tissue Resembles That in Normal Duodenal Tissue"

    Article Title: The Immune Cell Composition in Barrett's Metaplastic Tissue Resembles That in Normal Duodenal Tissue

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0033899

    Absence of IL-4 positive lymphocytes in BE cultures determined by intracellular FACS-staining. ( 1 ) Intracellular staining for IFN-γ and IL-4. Intracellular staining for IFN-γ and IL-4 in T-cells was determined after stimulation with PMA (20 ng/ml) and ionomycin (1 µM) for 6 hrs. Hereafter, CD4 + and CD8 + cells from ex-vivo cultures of BE and duodenum from BE (DUO BE) and duodenum from controls (DUO control) were analysed. Each bar represents mean value±SEM of the percentage of IFN-γ cells inside CD3 + CD4 + -population (panel A), percentage of IFN-γ cells inside CD3 + CD8 + -population (panel B), percentage of IL-4 cells inside CD3 + CD4 + -population (panel C) and percentage of IL-4 cells inside CD3 + CD8 + -population (panel D) (black bar: DUO control, dark grey bar: DUO BE, light grey bar: BE) ( ns not significant) (BE, n = 6,DUO BE, n = 5, DUO control, n = 4). ( 2 ) Representative flowcytometry plots for IFN-γ and IL-4 Representative flowcytometry plots of a representative staining of lymphocytes from a duodenal culture ex vivo . Gates for CD4 and CD8 were set and positive cells for IL-4 and IFN-γ were determined.
    Figure Legend Snippet: Absence of IL-4 positive lymphocytes in BE cultures determined by intracellular FACS-staining. ( 1 ) Intracellular staining for IFN-γ and IL-4. Intracellular staining for IFN-γ and IL-4 in T-cells was determined after stimulation with PMA (20 ng/ml) and ionomycin (1 µM) for 6 hrs. Hereafter, CD4 + and CD8 + cells from ex-vivo cultures of BE and duodenum from BE (DUO BE) and duodenum from controls (DUO control) were analysed. Each bar represents mean value±SEM of the percentage of IFN-γ cells inside CD3 + CD4 + -population (panel A), percentage of IFN-γ cells inside CD3 + CD8 + -population (panel B), percentage of IL-4 cells inside CD3 + CD4 + -population (panel C) and percentage of IL-4 cells inside CD3 + CD8 + -population (panel D) (black bar: DUO control, dark grey bar: DUO BE, light grey bar: BE) ( ns not significant) (BE, n = 6,DUO BE, n = 5, DUO control, n = 4). ( 2 ) Representative flowcytometry plots for IFN-γ and IL-4 Representative flowcytometry plots of a representative staining of lymphocytes from a duodenal culture ex vivo . Gates for CD4 and CD8 were set and positive cells for IL-4 and IFN-γ were determined.

    Techniques Used: FACS, Staining, Ex Vivo

    22) Product Images from "Variation in human cancer cell external phosphatidylserine is regulated by flippase activity and intracellular calcium"

    Article Title: Variation in human cancer cell external phosphatidylserine is regulated by flippase activity and intracellular calcium

    Journal: Oncotarget

    doi:

    Modulation of intracellular calcium by ionomycin or BAPTA-AM affects surface PS in human cancer cells A . Cells were treated with DMSO, ionomycin or BAPTA-AM, incubated with the calcium binding dye Fluo-4 Direct and fluorescence was measured using a microplate reader. Bars show relative changes in Fluo-4 Direct fluorescence signal for each cell line normalized to DMSO controls. B . Low and high surface PS cells were treated with DMSO or ionomycin and surface PS levels were assayed by staining with annexin V FITC, followed by flow cytometry. The axis indicates shift in the geometrical mean of annexin V FITC fluorescence in ionomycin treated vs untreated cells (U87ΔEGFR-Luc □, H1299 Δ, MDA-MB-231 ○, MDA-MB-231-Luc-D3H2LN ◆, Gli36 ▲, U373 ●, Mean—). (C) Low and and high surface PS cells were treated with DMSO or BAPTA-AM, followed by incubation with annexin V FITC and analyzed by flow cytometry. The axis indicates shift in the geometrical mean of annexin V FITC fluorescence in BAPTA-AM treated vs untreated cells (U87ΔEGFR-Luc □, H1299 Δ, MDA-MB-231 ○, MDA-MB-231-Luc-D3H2LN ◆, Gli36 ▲, U373 ●, Mean—).
    Figure Legend Snippet: Modulation of intracellular calcium by ionomycin or BAPTA-AM affects surface PS in human cancer cells A . Cells were treated with DMSO, ionomycin or BAPTA-AM, incubated with the calcium binding dye Fluo-4 Direct and fluorescence was measured using a microplate reader. Bars show relative changes in Fluo-4 Direct fluorescence signal for each cell line normalized to DMSO controls. B . Low and high surface PS cells were treated with DMSO or ionomycin and surface PS levels were assayed by staining with annexin V FITC, followed by flow cytometry. The axis indicates shift in the geometrical mean of annexin V FITC fluorescence in ionomycin treated vs untreated cells (U87ΔEGFR-Luc □, H1299 Δ, MDA-MB-231 ○, MDA-MB-231-Luc-D3H2LN ◆, Gli36 ▲, U373 ●, Mean—). (C) Low and and high surface PS cells were treated with DMSO or BAPTA-AM, followed by incubation with annexin V FITC and analyzed by flow cytometry. The axis indicates shift in the geometrical mean of annexin V FITC fluorescence in BAPTA-AM treated vs untreated cells (U87ΔEGFR-Luc □, H1299 Δ, MDA-MB-231 ○, MDA-MB-231-Luc-D3H2LN ◆, Gli36 ▲, U373 ●, Mean—).

    Techniques Used: Incubation, Binding Assay, Fluorescence, Staining, Flow Cytometry, Cytometry, Multiple Displacement Amplification

    23) Product Images from "Shared and Unique Features Distinguishing Follicular T Helper and Regulatory Cells of Peripheral Lymph Node and Peyer’s Patches"

    Article Title: Shared and Unique Features Distinguishing Follicular T Helper and Regulatory Cells of Peripheral Lymph Node and Peyer’s Patches

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.00714

    Cytokine profiles of TFH and TFR cells from Peyer’s Patches (PP) and peripheral lymph node (pLN). Total cell preparations from PP and pLNs d6, d12, and d25 p.i. were in vitro stimulated with PMA/ionomycin in the presence of Brefeldin A, followed by intracellular detection of the indicated cytokines by flow cytometry. (A) Representative plots depicting expression of IL4, IL10, IL17, and IFNγ by TFH and TFR cells of PP or pLN 12 days p.i. as indicated are shown. (B) Summary of (A) (shown are mean ± SD). (C) 3D representation of time kinetics of cytokine production by pLN TFH and TFR cells from d6, d12, and d25 p.i. with KLH + Alum. Data from PP cells are also shown. Frequencies of positive cells are measured via intracellular detection of cytokines by flow cytometry as in (A) . Data were combined from two independent experiments with two mice per experiment for each time point. Shown are mean values.
    Figure Legend Snippet: Cytokine profiles of TFH and TFR cells from Peyer’s Patches (PP) and peripheral lymph node (pLN). Total cell preparations from PP and pLNs d6, d12, and d25 p.i. were in vitro stimulated with PMA/ionomycin in the presence of Brefeldin A, followed by intracellular detection of the indicated cytokines by flow cytometry. (A) Representative plots depicting expression of IL4, IL10, IL17, and IFNγ by TFH and TFR cells of PP or pLN 12 days p.i. as indicated are shown. (B) Summary of (A) (shown are mean ± SD). (C) 3D representation of time kinetics of cytokine production by pLN TFH and TFR cells from d6, d12, and d25 p.i. with KLH + Alum. Data from PP cells are also shown. Frequencies of positive cells are measured via intracellular detection of cytokines by flow cytometry as in (A) . Data were combined from two independent experiments with two mice per experiment for each time point. Shown are mean values.

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

    24) Product Images from "Herpes Simplex Virus Type 2 Glycoprotein H Interacts with Integrin αvβ3 To Facilitate Viral Entry and Calcium Signaling in Human Genital Tract Epithelial Cells"

    Article Title: Herpes Simplex Virus Type 2 Glycoprotein H Interacts with Integrin αvβ3 To Facilitate Viral Entry and Calcium Signaling in Human Genital Tract Epithelial Cells

    Journal: Journal of Virology

    doi: 10.1128/JVI.00725-14

    Integrin αvβ3 contributes to the virus-induced cytoplasmic Ca 2+ response post-Akt phosphorylation. (a) CaSki cells transfected with siIntegrinαvβ3 RNA were mock infected (serum free medium) or infected with purified HSV-2(G) (10 PFU/cell), and cell lysates were prepared for Western blot analysis at the indicated times p.i. Blots were incubated with anti pS 473 -Akt123 and then stripped and probed with anti-total Akt123. A representative blot from 3 independent experiments is shown. (b) CaSki cells were loaded with Calcium Green 72 h posttransfection with the indicated siRNA and synchronously mock infected or infected with purified HSV-2(G) (5 PFU/cell). Live images were acquired 3 min after a temperature shift to 37°C. Cellular membranes were stained with CellTrace (red), nuclei were stained with Hoechst (blue), and Ca 2+ is green. Representative XYZ images from 3 independent experiments are shown. Bars = 9.2 μm. (c) Transfected CaSki cells were loaded with fura-2, infected with purified HSV-2(G) (2 PFU/cell), or mock infected. To assess whether siRNA transfections impacted the intracellular Ca 2+ stores, uninfected cells were treated with 1 μM ionomycin. The mean intracellular Ca 2+ concentration (nM) over 1 h was calculated from 4 wells, each containing 5 × 10 4 cells; the asterisk indicates a significant increase in Ca 2+ concentration relative to the mock-infected control ( P
    Figure Legend Snippet: Integrin αvβ3 contributes to the virus-induced cytoplasmic Ca 2+ response post-Akt phosphorylation. (a) CaSki cells transfected with siIntegrinαvβ3 RNA were mock infected (serum free medium) or infected with purified HSV-2(G) (10 PFU/cell), and cell lysates were prepared for Western blot analysis at the indicated times p.i. Blots were incubated with anti pS 473 -Akt123 and then stripped and probed with anti-total Akt123. A representative blot from 3 independent experiments is shown. (b) CaSki cells were loaded with Calcium Green 72 h posttransfection with the indicated siRNA and synchronously mock infected or infected with purified HSV-2(G) (5 PFU/cell). Live images were acquired 3 min after a temperature shift to 37°C. Cellular membranes were stained with CellTrace (red), nuclei were stained with Hoechst (blue), and Ca 2+ is green. Representative XYZ images from 3 independent experiments are shown. Bars = 9.2 μm. (c) Transfected CaSki cells were loaded with fura-2, infected with purified HSV-2(G) (2 PFU/cell), or mock infected. To assess whether siRNA transfections impacted the intracellular Ca 2+ stores, uninfected cells were treated with 1 μM ionomycin. The mean intracellular Ca 2+ concentration (nM) over 1 h was calculated from 4 wells, each containing 5 × 10 4 cells; the asterisk indicates a significant increase in Ca 2+ concentration relative to the mock-infected control ( P

    Techniques Used: Transfection, Infection, Purification, Western Blot, Incubation, Staining, Concentration Assay

    25) Product Images from ""

    Article Title:

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.304329

    Stimulation of T cell blasts leads to a mobilization but not stabilization of CTLA-4 at the cell surface. A , human CD4+ T cell blasts were generated using anti-CD3 anti-CD28 beads for 4 days. Cells were then isolated and re-stimulated with PMA+ ionomycin or fresh anti-CD3 anti-CD28 beads for 3 h at 37 °C in the presence of anti-CTLA-4 PE before analysis by flow cytometry. B , T cell blasts were re-stimulated as in A in the presence of anti-CTLA-4 PE or anti-CD28 PE at 37 °C. Cells were then placed on ice, and the remaining surface receptors were labeled by incubation with Alexa647-conjugated anti-mouse secondary antibody. Cells were analyzed by flow cytometry. The boxed area indicates an increase in CTLA-4 labeling due to stimulation. C , the ratio of surface to labeled fluorescence for cells labeled in B is plotted. D , cells stained for CTLA-4 as in B were fixed and then visualized by confocal microscopy for 37 °C staining ( red ) and surface staining ( blue ).
    Figure Legend Snippet: Stimulation of T cell blasts leads to a mobilization but not stabilization of CTLA-4 at the cell surface. A , human CD4+ T cell blasts were generated using anti-CD3 anti-CD28 beads for 4 days. Cells were then isolated and re-stimulated with PMA+ ionomycin or fresh anti-CD3 anti-CD28 beads for 3 h at 37 °C in the presence of anti-CTLA-4 PE before analysis by flow cytometry. B , T cell blasts were re-stimulated as in A in the presence of anti-CTLA-4 PE or anti-CD28 PE at 37 °C. Cells were then placed on ice, and the remaining surface receptors were labeled by incubation with Alexa647-conjugated anti-mouse secondary antibody. Cells were analyzed by flow cytometry. The boxed area indicates an increase in CTLA-4 labeling due to stimulation. C , the ratio of surface to labeled fluorescence for cells labeled in B is plotted. D , cells stained for CTLA-4 as in B were fixed and then visualized by confocal microscopy for 37 °C staining ( red ) and surface staining ( blue ).

    Techniques Used: Generated, Isolation, Flow Cytometry, Cytometry, Labeling, Incubation, Fluorescence, Staining, Confocal Microscopy

    Stimulation increases trafficking of CTLA-4 in the absence of increased synthesis. A , CTLA-4-expressing Jurkat cells were labeled at 37 °C with anti-CTLA-4 PE for 1 h after stimulation with PMA/ionomycin and analyzed by flow cytometry. B , Jurkat cells were labeled at 37 °C with anti-CTLA-4 PE in the presence or absence of PMA/ionomycin for 60 min ( cycling CTLA-4 ). Cells were then fixed, and total CTLA-4 was stained with a goat anti-CTLA-4 C-terminal antibody followed by Alexa633 anti-goat secondary and analyzed by flow cytometry. C , shown is the ratio of labeled to total CTLA-4 for cells, stained as in B. D , Jurkat cells were labeled at 37 °C with anti-CTLA-4 PE in the presence or absence of PMA/ionomycin for 60 min (cycling CTLA-4). Cells were then placed on ice, and the remaining surface receptors were labeled by incubation with Alexa647-conjugated anti-mouse secondary antibody (surface CTLA-4). Cells were analyzed by flow cytometry. E , shown is the ratio of surface to cycling CTLA-4 fluorescence for cells labeled in D .
    Figure Legend Snippet: Stimulation increases trafficking of CTLA-4 in the absence of increased synthesis. A , CTLA-4-expressing Jurkat cells were labeled at 37 °C with anti-CTLA-4 PE for 1 h after stimulation with PMA/ionomycin and analyzed by flow cytometry. B , Jurkat cells were labeled at 37 °C with anti-CTLA-4 PE in the presence or absence of PMA/ionomycin for 60 min ( cycling CTLA-4 ). Cells were then fixed, and total CTLA-4 was stained with a goat anti-CTLA-4 C-terminal antibody followed by Alexa633 anti-goat secondary and analyzed by flow cytometry. C , shown is the ratio of labeled to total CTLA-4 for cells, stained as in B. D , Jurkat cells were labeled at 37 °C with anti-CTLA-4 PE in the presence or absence of PMA/ionomycin for 60 min (cycling CTLA-4). Cells were then placed on ice, and the remaining surface receptors were labeled by incubation with Alexa647-conjugated anti-mouse secondary antibody (surface CTLA-4). Cells were analyzed by flow cytometry. E , shown is the ratio of surface to cycling CTLA-4 fluorescence for cells labeled in D .

    Techniques Used: Expressing, Labeling, Flow Cytometry, Cytometry, Staining, Incubation, Fluorescence

    26) Product Images from "Targeting the NFAT1-MDM2-MDMX Network Inhibits the Proliferation and Invasion of Prostate Cancer Cells, Independent of p53 and Androgen"

    Article Title: Targeting the NFAT1-MDM2-MDMX Network Inhibits the Proliferation and Invasion of Prostate Cancer Cells, Independent of p53 and Androgen

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2017.00917

    Inulanolide A inhibits NFAT1-mediated MDM2 transcription. (A) LNCaP and PC3 cells were exposed to InuA (5 μM) for 24 h in the presence or absence of ionomycin (ION). Nuclear proteins were extracted and incubated with an MDM2 probe, followed by an EMSA assay. NE, nuclear extract. (B) LNCaP and PC3 cells were exposed to InuA (5 μM) for 24 h. The crosslinked chromatin was immunoprecipitated with anti-NFAT1 or IgG antibodies, followed by a real-time PCR analysis. (C) LNCaP and PC3 cells were transfected with NFAT1 siRNA or the respective control siRNA for 36 h, followed by exposure to InuA at the indicated concentrations for 24 h. The relative MDM2 mRNA levels and the protein levels of NFAT1 and MDM2 were determined by quantitative real-time PCR and Western blotting, respectively. (D) LNCaP and PC3 cells were co-transfected with DN-NFAT and MDM2 P2 promoter luciferase for 24 h, followed by exposure to InuA at the indicated concentrations for 24 h. The relative luciferase levels were then determined using a dual-reporter gene detection system. The protein levels of DN-NFAT and MDM2 were determined by a Western blot analysis. Data are representative of at least three experiments ( ∗ P
    Figure Legend Snippet: Inulanolide A inhibits NFAT1-mediated MDM2 transcription. (A) LNCaP and PC3 cells were exposed to InuA (5 μM) for 24 h in the presence or absence of ionomycin (ION). Nuclear proteins were extracted and incubated with an MDM2 probe, followed by an EMSA assay. NE, nuclear extract. (B) LNCaP and PC3 cells were exposed to InuA (5 μM) for 24 h. The crosslinked chromatin was immunoprecipitated with anti-NFAT1 or IgG antibodies, followed by a real-time PCR analysis. (C) LNCaP and PC3 cells were transfected with NFAT1 siRNA or the respective control siRNA for 36 h, followed by exposure to InuA at the indicated concentrations for 24 h. The relative MDM2 mRNA levels and the protein levels of NFAT1 and MDM2 were determined by quantitative real-time PCR and Western blotting, respectively. (D) LNCaP and PC3 cells were co-transfected with DN-NFAT and MDM2 P2 promoter luciferase for 24 h, followed by exposure to InuA at the indicated concentrations for 24 h. The relative luciferase levels were then determined using a dual-reporter gene detection system. The protein levels of DN-NFAT and MDM2 were determined by a Western blot analysis. Data are representative of at least three experiments ( ∗ P

    Techniques Used: Incubation, Immunoprecipitation, Real-time Polymerase Chain Reaction, Transfection, Western Blot, Luciferase

    Inulanolide A promotes NFAT1 protein degradation and inhibits NFAT1 nuclear translation. (A) LNCaP and PC3 cells were treated with InuA (2 μM) for 24 h, followed by exposure to a protein synthesis inhibitor, cycloheximide (CHX, 15 μg/mL). The protein expression levels of NFAT1 were detected by Western blotting at the indicated times after exposure to CHX. Graphs (on the right) show the quantification of the immunoblotting data. (B) LNCaP and PC3 cells were co-transfected with NFAT1 and ubiquitin plasmids, followed by treatment with InuA at the indicated concentrations for 24 h. Cell lysates were subjected to immunoprecipitation with an anti-NFAT1 antibody. The ubiquitinated NFAT1 was detected using an anti-ubiquitin antibody. (C) LNCaP and PC3 cells were treated with InuA (2 μM) in the presence or absence of ionomycin (ION; 4 μM) or cyclosporine A (CsA; 2 μM) for 24 h. The nuclear and cytosolic proteins were extracted and examined by Western blotting. Lamin B and α-tubulin were, respectively, used as the internal references for the nuclear and cytosolic extracts. Data are representative of three or more experiments.
    Figure Legend Snippet: Inulanolide A promotes NFAT1 protein degradation and inhibits NFAT1 nuclear translation. (A) LNCaP and PC3 cells were treated with InuA (2 μM) for 24 h, followed by exposure to a protein synthesis inhibitor, cycloheximide (CHX, 15 μg/mL). The protein expression levels of NFAT1 were detected by Western blotting at the indicated times after exposure to CHX. Graphs (on the right) show the quantification of the immunoblotting data. (B) LNCaP and PC3 cells were co-transfected with NFAT1 and ubiquitin plasmids, followed by treatment with InuA at the indicated concentrations for 24 h. Cell lysates were subjected to immunoprecipitation with an anti-NFAT1 antibody. The ubiquitinated NFAT1 was detected using an anti-ubiquitin antibody. (C) LNCaP and PC3 cells were treated with InuA (2 μM) in the presence or absence of ionomycin (ION; 4 μM) or cyclosporine A (CsA; 2 μM) for 24 h. The nuclear and cytosolic proteins were extracted and examined by Western blotting. Lamin B and α-tubulin were, respectively, used as the internal references for the nuclear and cytosolic extracts. Data are representative of three or more experiments.

    Techniques Used: Expressing, Western Blot, Transfection, Immunoprecipitation

    27) Product Images from "RCAN-11R peptide provides immunosuppression for fully mismatched islet allografts in mice"

    Article Title: RCAN-11R peptide provides immunosuppression for fully mismatched islet allografts in mice

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-02934-3

    The dose-response and half-life of RCAN-11R. ( a ) The dose-response of RCAN-11R. Jurkat cells were treated with 0.1–100 μM RCAN-11R or 1 μM FK506 for 1 h, then with 200 nM PMA and 4 μM ionomycin for an additional 12 h. The cells were subjected to a quantitative RT-PCR. ( b ) Dephosphorylation of NFAT. Jurkat cells were treated with 0.1, 1, or 10 μM RCAN-11R for 1 h and with 20 nM PMA and 300 nM ionomycin for an additional 15 min and then western blot was performed. ( c ) The half-life of RCAN-11R. Jurkat cells were treated with 20 μM RCAN-11R for periods of 1–72 h or with 1 μM FK506 for 1 h, then with 200 nM PMA and 4 μM ionomycin for an additional 12 h. The cells were then subjected to a quantitative RT-PCR. The values represent the mean ± SE of five independent experiments.
    Figure Legend Snippet: The dose-response and half-life of RCAN-11R. ( a ) The dose-response of RCAN-11R. Jurkat cells were treated with 0.1–100 μM RCAN-11R or 1 μM FK506 for 1 h, then with 200 nM PMA and 4 μM ionomycin for an additional 12 h. The cells were subjected to a quantitative RT-PCR. ( b ) Dephosphorylation of NFAT. Jurkat cells were treated with 0.1, 1, or 10 μM RCAN-11R for 1 h and with 20 nM PMA and 300 nM ionomycin for an additional 15 min and then western blot was performed. ( c ) The half-life of RCAN-11R. Jurkat cells were treated with 20 μM RCAN-11R for periods of 1–72 h or with 1 μM FK506 for 1 h, then with 200 nM PMA and 4 μM ionomycin for an additional 12 h. The cells were then subjected to a quantitative RT-PCR. The values represent the mean ± SE of five independent experiments.

    Techniques Used: Quantitative RT-PCR, De-Phosphorylation Assay, Western Blot

    The transduction of RCAN-11R into lymphocytes and the inhibition of NFAT nuclear translocation. ( a ) The CIC (Calcineurin inhibitor of Calcipressin) motif of the human RCAN protein family (upper schematic illustration) and the sequence of RCAN-11R and the scramble RCAN-11R peptides (lower schematic illustration). ( b ) Transduction of RCAN-11R into lymphocytes. Jurkat cells were incubated with 10 μM FITC-RCAN-11R and examined using an Olympus confocal microscope. Scale bar = 100 µm. ( c ) The inhibition of NFAT nuclear translocation. HEK 293 cells that had been transfected with GFP-NFAT-1 plasmid were incubated with 1 μM FK506 or 20 μM RCAN-11R/scRCAN-11R for 2 h and then 500 nM ionomycin was added to the culture medium. Scale bar = 20 µm. ( d ) The percentage of NFAT nuclear translocation. GFP-NFAT-1 nuclear translocation was quantified by microscopy. *p
    Figure Legend Snippet: The transduction of RCAN-11R into lymphocytes and the inhibition of NFAT nuclear translocation. ( a ) The CIC (Calcineurin inhibitor of Calcipressin) motif of the human RCAN protein family (upper schematic illustration) and the sequence of RCAN-11R and the scramble RCAN-11R peptides (lower schematic illustration). ( b ) Transduction of RCAN-11R into lymphocytes. Jurkat cells were incubated with 10 μM FITC-RCAN-11R and examined using an Olympus confocal microscope. Scale bar = 100 µm. ( c ) The inhibition of NFAT nuclear translocation. HEK 293 cells that had been transfected with GFP-NFAT-1 plasmid were incubated with 1 μM FK506 or 20 μM RCAN-11R/scRCAN-11R for 2 h and then 500 nM ionomycin was added to the culture medium. Scale bar = 20 µm. ( d ) The percentage of NFAT nuclear translocation. GFP-NFAT-1 nuclear translocation was quantified by microscopy. *p

    Techniques Used: Transduction, Inhibition, Translocation Assay, Sequencing, Incubation, Microscopy, Transfection, Plasmid Preparation

    The inhibition of NFAT reporter activity and the production of IL2. ( a , b ) NFAT ( a ) and NF-κB ( b ) reporter activity. Jurkat cells were electroporated with 5 μg of pNFAT-SEAP or pNF-κB-SEAP, respectively. The cells were incubated with 1 μM FK506 or 20 μM RCAN-11R/scRCAN-11R for 1 h, and stimulated with 200 nM PMA and 4 μM ionomycin for 12 h. ( c ) The inhibition of IL-2 transcription. Jurkat cells were treated with 1 μM FK506 or 20 μM RCAN-11R/scRCAN-11R for 1 h, then with 200 nM PMA and 4 μM ionomycin for an additional 12 h. The cells were subjected to a quantitative RT-PCR. ( d ) The inhibition of IL-2 production. Jurkat cells were treated with 1 μM FK506 or 20 μM RCAN-11R/scRCAN-11R for 1 h and then incubated with 200 nM PMA and 4 μM ionomycin for an additional 12 h. *p
    Figure Legend Snippet: The inhibition of NFAT reporter activity and the production of IL2. ( a , b ) NFAT ( a ) and NF-κB ( b ) reporter activity. Jurkat cells were electroporated with 5 μg of pNFAT-SEAP or pNF-κB-SEAP, respectively. The cells were incubated with 1 μM FK506 or 20 μM RCAN-11R/scRCAN-11R for 1 h, and stimulated with 200 nM PMA and 4 μM ionomycin for 12 h. ( c ) The inhibition of IL-2 transcription. Jurkat cells were treated with 1 μM FK506 or 20 μM RCAN-11R/scRCAN-11R for 1 h, then with 200 nM PMA and 4 μM ionomycin for an additional 12 h. The cells were subjected to a quantitative RT-PCR. ( d ) The inhibition of IL-2 production. Jurkat cells were treated with 1 μM FK506 or 20 μM RCAN-11R/scRCAN-11R for 1 h and then incubated with 200 nM PMA and 4 μM ionomycin for an additional 12 h. *p

    Techniques Used: Inhibition, Activity Assay, Incubation, Quantitative RT-PCR

    28) Product Images from "Distinct Transcriptional and Alternative Splicing Signatures of Decidual CD4+ T Cells in Early Human Pregnancy"

    Article Title: Distinct Transcriptional and Alternative Splicing Signatures of Decidual CD4+ T Cells in Early Human Pregnancy

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2017.00682

    dCD4 T cells stay in M phase and show increased activation, proliferation, and cytokine production, as well as contain Th1, Th17, and Treg cell subsets and display an effector-memory phenotype. (A–D) GSEA plots of GO categories, including M phase of mitotic cell cycle, mitotic sister chromatin segregation (A) ; lymphocyte activation, T cell activation (B) ; cell proliferation, positive regulation of cell proliferation (C) ; and cytokine production and cytokine secretion (D) . (E,G,I,L) Comparison of the gene expression (measured as the read count) of IFNG, IL17A, IL4 , and FOXP3 between in paired pCD4 and dCD4 T cells at rest. Each symbol reflects a sample and each line reflects the samples from an individual ( n = 3 per group). (F,H,J,K) Comparison of the expression of IFN-γ, IL-17A, IL-4, and Foxp3 between paired pCD4 and dCD4 T cells as determined by intracellular staining upon stimulation with phorbol-12-myristate-13-acetate and ionomycin in the presence of brefeldin and monensin. Similar results were obtained from four individuals at the first trimester of normal pregnancy. (M–O) Representative flow cytometric plots (M) , bar graphs (N) and pie charts (O) displaying the proportions of native (T N , CD45RO − CCR7 + ), effector (T E , CD45RO − CCR7 − ), T CM (CD45RO + CCR7 + ), and T EM (CD45RO + CCR7 − ) cells in paired pCD4 and dCD4 T cells. Each symbol reflects a sample and each line reflects the samples from the same person ( n = 4 per group). pCD4 T, peripheral blood CD4 + T; dCD4 T, decidual CD4 + T; GO, Gene Ontology; GSEA, gene set enrichment analysis; Nom, Nominal; NES, Normalized Enrichment Score; UD, undetected.
    Figure Legend Snippet: dCD4 T cells stay in M phase and show increased activation, proliferation, and cytokine production, as well as contain Th1, Th17, and Treg cell subsets and display an effector-memory phenotype. (A–D) GSEA plots of GO categories, including M phase of mitotic cell cycle, mitotic sister chromatin segregation (A) ; lymphocyte activation, T cell activation (B) ; cell proliferation, positive regulation of cell proliferation (C) ; and cytokine production and cytokine secretion (D) . (E,G,I,L) Comparison of the gene expression (measured as the read count) of IFNG, IL17A, IL4 , and FOXP3 between in paired pCD4 and dCD4 T cells at rest. Each symbol reflects a sample and each line reflects the samples from an individual ( n = 3 per group). (F,H,J,K) Comparison of the expression of IFN-γ, IL-17A, IL-4, and Foxp3 between paired pCD4 and dCD4 T cells as determined by intracellular staining upon stimulation with phorbol-12-myristate-13-acetate and ionomycin in the presence of brefeldin and monensin. Similar results were obtained from four individuals at the first trimester of normal pregnancy. (M–O) Representative flow cytometric plots (M) , bar graphs (N) and pie charts (O) displaying the proportions of native (T N , CD45RO − CCR7 + ), effector (T E , CD45RO − CCR7 − ), T CM (CD45RO + CCR7 + ), and T EM (CD45RO + CCR7 − ) cells in paired pCD4 and dCD4 T cells. Each symbol reflects a sample and each line reflects the samples from the same person ( n = 4 per group). pCD4 T, peripheral blood CD4 + T; dCD4 T, decidual CD4 + T; GO, Gene Ontology; GSEA, gene set enrichment analysis; Nom, Nominal; NES, Normalized Enrichment Score; UD, undetected.

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

    29) Product Images from "Intestinal Microbiota-Derived GABA Mediates Interleukin-17 Expression during Enterotoxigenic Escherichia coli Infection"

    Article Title: Intestinal Microbiota-Derived GABA Mediates Interleukin-17 Expression during Enterotoxigenic Escherichia coli Infection

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2016.00685

    Enterotoxigenic Escherichia coli (ETEC) induces intestinal interleukin-17 (IL-17) expression . (A) Relative mRNA expression of indicated cytokines in piglet jejunum samples ( n = 6). (B) Relative mRNA expression of IL-17 in mouse jejunum. (C) Relative mRNA expression of IL-17 in mouse ileum. (D) Nuclear and cytoplasmic abundance of RAR-related orphan receptor gamma t (RORγt) in mouse jejunum samples. In this, and in all subsequent figures, the statistical data (mean ± SEM; n = 5) below the image indicate the relative band amount of indicated protein obtained by dividing the actin or proliferating cell nuclear antigen (PCNA) band intensity by the indicated band intensity in each lane. (E) Nuclear and cytoplasmic abundance of RORγt in mouse ileum samples. (F,H,I) ICR mice were infected with ETEC for 6 h, and lymphocytes from the jejunal lamina propria were isolated and stimulated for 5 h with PMA, ionomycin, and monensin, followed by flow cytometry analysis of the frequency of CD3 + IL-17 + cells (F) , CD3 + CD4 + IL-17 + cells (H) , and CD3 + TCR γδ + IL-17 + cells (I) . (G) Relative mRNA expression of IL-17 in Rag1 −/− mouse jejunum at 6-h post-ETEC infection. (J) Relative mRNA expression of IL-17 in TCR delta −/− mouse jejunum at 6-h post-ETEC infection. *indicates a statistically significant difference between two groups ( p
    Figure Legend Snippet: Enterotoxigenic Escherichia coli (ETEC) induces intestinal interleukin-17 (IL-17) expression . (A) Relative mRNA expression of indicated cytokines in piglet jejunum samples ( n = 6). (B) Relative mRNA expression of IL-17 in mouse jejunum. (C) Relative mRNA expression of IL-17 in mouse ileum. (D) Nuclear and cytoplasmic abundance of RAR-related orphan receptor gamma t (RORγt) in mouse jejunum samples. In this, and in all subsequent figures, the statistical data (mean ± SEM; n = 5) below the image indicate the relative band amount of indicated protein obtained by dividing the actin or proliferating cell nuclear antigen (PCNA) band intensity by the indicated band intensity in each lane. (E) Nuclear and cytoplasmic abundance of RORγt in mouse ileum samples. (F,H,I) ICR mice were infected with ETEC for 6 h, and lymphocytes from the jejunal lamina propria were isolated and stimulated for 5 h with PMA, ionomycin, and monensin, followed by flow cytometry analysis of the frequency of CD3 + IL-17 + cells (F) , CD3 + CD4 + IL-17 + cells (H) , and CD3 + TCR γδ + IL-17 + cells (I) . (G) Relative mRNA expression of IL-17 in Rag1 −/− mouse jejunum at 6-h post-ETEC infection. (J) Relative mRNA expression of IL-17 in TCR delta −/− mouse jejunum at 6-h post-ETEC infection. *indicates a statistically significant difference between two groups ( p

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

    30) Product Images from "CD4+ T Cells of Myasthenia Gravis Patients Are Characterized by Increased IL-21, IL-4, and IL-17A Productions and Higher Presence of PD-1 and ICOS"

    Article Title: CD4+ T Cells of Myasthenia Gravis Patients Are Characterized by Increased IL-21, IL-4, and IL-17A Productions and Higher Presence of PD-1 and ICOS

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2020.00809

    Cytokine production of CD4 + T cells in myasthenia gravis (MG) subgroups. (A) Measurement of intracellular IL-21, IL-4, IL-17A, IL-10, and IFN-γ in CD4 + T cells of a patient and a healthy control (HC) by flow cytometry after 4 h of stimulation with phorbol 12-myristate 13-acetate and ionomycin in cell culture. (B) The AChR-MG ( n = 54) patients had higher IL-21, IL-4, IL-17A, and IL-10 ( p
    Figure Legend Snippet: Cytokine production of CD4 + T cells in myasthenia gravis (MG) subgroups. (A) Measurement of intracellular IL-21, IL-4, IL-17A, IL-10, and IFN-γ in CD4 + T cells of a patient and a healthy control (HC) by flow cytometry after 4 h of stimulation with phorbol 12-myristate 13-acetate and ionomycin in cell culture. (B) The AChR-MG ( n = 54) patients had higher IL-21, IL-4, IL-17A, and IL-10 ( p

    Techniques Used: Flow Cytometry, Cell Culture

    31) Product Images from "Oxidized Phospholipid OxPAPC Activates TRPA1 and Contributes to Chronic Inflammatory Pain in Mice"

    Article Title: Oxidized Phospholipid OxPAPC Activates TRPA1 and Contributes to Chronic Inflammatory Pain in Mice

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0165200

    OxPAPC specifically activates human TRPA1 (hTRPA1) channels expressed in HEK293 cells. (A) Representative images derived from Fura-2 ratiometric analysis showing [Ca 2+ ] i changes in HEK293 cells transfected with hTRPA1 cDNA or empty vector pCDNA3.1 in response to OxPAPC (10 μg/ml) and mustard oil (MO, 70 μM). (B) Averaged Ca 2+ responses from OxPAPC experiments in panel (A). At the end of the experiments, ionomycin (1.5 μM) was applied to activate all viable cells in the field. n > 40 cells/group. (C) Comparison of averaged Ca 2+ responses induced by 0, 10, 30 or 100 μg/ml OxPAPC. Imaging traces are overlaid for comparison. n > 40 cells/group. (D) Dose-response analysis of OxPAPC activation of calcium influx in hTRPA1 transfected cells with vector-transfected cells as controls. The responses of OxPAPC were normalized to those of ionomycin. EC 50 = 9.5 μg/ml. Each data point represents 5–6 separate tests. (E) Effects of OxPAPC (10 μg/ml) on hTRPA1 compared to human TRPV1, TRPV4, TRPM8 and empty vector in Ca 2+ imaging tests. The dotted line shows the basal level obtained from cells transfected with empty vector alone. (F) Comparison of the effects of different doses of OxPAPC, PAPC and DMPC on hTRPA1 by Ca 2+ imaging. Responses were normalized to ionomycin applied at the end of the tests. 10, 30 and 100 μg/ml of each lipid product were tested and compared. n = 6 tests/group. *p
    Figure Legend Snippet: OxPAPC specifically activates human TRPA1 (hTRPA1) channels expressed in HEK293 cells. (A) Representative images derived from Fura-2 ratiometric analysis showing [Ca 2+ ] i changes in HEK293 cells transfected with hTRPA1 cDNA or empty vector pCDNA3.1 in response to OxPAPC (10 μg/ml) and mustard oil (MO, 70 μM). (B) Averaged Ca 2+ responses from OxPAPC experiments in panel (A). At the end of the experiments, ionomycin (1.5 μM) was applied to activate all viable cells in the field. n > 40 cells/group. (C) Comparison of averaged Ca 2+ responses induced by 0, 10, 30 or 100 μg/ml OxPAPC. Imaging traces are overlaid for comparison. n > 40 cells/group. (D) Dose-response analysis of OxPAPC activation of calcium influx in hTRPA1 transfected cells with vector-transfected cells as controls. The responses of OxPAPC were normalized to those of ionomycin. EC 50 = 9.5 μg/ml. Each data point represents 5–6 separate tests. (E) Effects of OxPAPC (10 μg/ml) on hTRPA1 compared to human TRPV1, TRPV4, TRPM8 and empty vector in Ca 2+ imaging tests. The dotted line shows the basal level obtained from cells transfected with empty vector alone. (F) Comparison of the effects of different doses of OxPAPC, PAPC and DMPC on hTRPA1 by Ca 2+ imaging. Responses were normalized to ionomycin applied at the end of the tests. 10, 30 and 100 μg/ml of each lipid product were tested and compared. n = 6 tests/group. *p

    Techniques Used: Derivative Assay, Transfection, Plasmid Preparation, Imaging, Activation Assay

    OxPAPC-induced TRPA1 activation is independent of EP2 and DP receptors in both HEK293 cells and native DRG neurons. (A) Summary of OxPAPC-induced Ca 2+ responses in HEK293 cells expressing hTRPA1. Cells were first superfused with vehicle only (0.05% DMSO), AH6809 (10 μM), PF04418948 (20 nM) or ruthenium red (10 μM) for 5 min before recording started and then recorded in the continued presence of above treatments. Cells were challenged with OxPAPC (30 μM) and subsequently with mustard oil (MO, 70 μM) and ionomycin (1 μM). Responses of > 50 cells were averaged from each group. (B) Average peak amplitudes of OxPAPC-induced Ca 2+ responses in HEK293 cells as shown in (A). (C) Percentages of mouse DRG neurons responding to OxPAPC in vehicle only, or in the presence of AH6809 (10 μM) or PF04418948 (20 nM). Neurons were pretreated with the antagonists for 5 min before recording and recorded in the continued presence of the treatments. Cells were challenged with OxPAPC (10 μM) for 100 s and subsequently with KCl (40 mM) for 40 s. n = 5–8 tests/group, averages from 100–200 neurons per group. (D) Maximal Fura-2 emission ratio amplitudes of OxPAPC-induced Ca 2+ responses in mouse DRG neurons recorded in (C). **p
    Figure Legend Snippet: OxPAPC-induced TRPA1 activation is independent of EP2 and DP receptors in both HEK293 cells and native DRG neurons. (A) Summary of OxPAPC-induced Ca 2+ responses in HEK293 cells expressing hTRPA1. Cells were first superfused with vehicle only (0.05% DMSO), AH6809 (10 μM), PF04418948 (20 nM) or ruthenium red (10 μM) for 5 min before recording started and then recorded in the continued presence of above treatments. Cells were challenged with OxPAPC (30 μM) and subsequently with mustard oil (MO, 70 μM) and ionomycin (1 μM). Responses of > 50 cells were averaged from each group. (B) Average peak amplitudes of OxPAPC-induced Ca 2+ responses in HEK293 cells as shown in (A). (C) Percentages of mouse DRG neurons responding to OxPAPC in vehicle only, or in the presence of AH6809 (10 μM) or PF04418948 (20 nM). Neurons were pretreated with the antagonists for 5 min before recording and recorded in the continued presence of the treatments. Cells were challenged with OxPAPC (10 μM) for 100 s and subsequently with KCl (40 mM) for 40 s. n = 5–8 tests/group, averages from 100–200 neurons per group. (D) Maximal Fura-2 emission ratio amplitudes of OxPAPC-induced Ca 2+ responses in mouse DRG neurons recorded in (C). **p

    Techniques Used: Activation Assay, Expressing

    32) Product Images from "Reduced Leukocyte Infiltration in Absence of Eosinophils Correlates with Decreased Tissue Damage and Disease Susceptibility in ΔdblGATA Mice during Murine Neurocysticercosis"

    Article Title: Reduced Leukocyte Infiltration in Absence of Eosinophils Correlates with Decreased Tissue Damage and Disease Susceptibility in ΔdblGATA Mice during Murine Neurocysticercosis

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0004787

    Eosinophil-deficient mice have reduced Th2 response during murine NCC. Immunofluorescent staining for IL-4 shows negative signals in mock-infected WT ( A ) and ΔdblGATA brains ( B ). However, IL-4 is upregulated after infection and abundant in inflammatory infiltrates of WT mice ( C ) and to a lesser extent in ΔdblGATA mice ( D, E ). Total RNA isolated from enriched brain T cells was subjected to reverse transcription and transcript levels of IL-4 and Gata3 assessed by qRT-PCR and results shown as fold change over values from ΔdblGATA mice ( F ) revealed an enrichment of both transcripts in WT T cells. CD4 T cells were isolated from infected brains and incubated with PMA/Ionomycin and Brefeldin A/Monensin followed by intracellular IL-4 staining ( G, H ). The proportion of CD4 + IL-4 + T cells ( I ) was increased in T cells isolated from infected WT mice.
    Figure Legend Snippet: Eosinophil-deficient mice have reduced Th2 response during murine NCC. Immunofluorescent staining for IL-4 shows negative signals in mock-infected WT ( A ) and ΔdblGATA brains ( B ). However, IL-4 is upregulated after infection and abundant in inflammatory infiltrates of WT mice ( C ) and to a lesser extent in ΔdblGATA mice ( D, E ). Total RNA isolated from enriched brain T cells was subjected to reverse transcription and transcript levels of IL-4 and Gata3 assessed by qRT-PCR and results shown as fold change over values from ΔdblGATA mice ( F ) revealed an enrichment of both transcripts in WT T cells. CD4 T cells were isolated from infected brains and incubated with PMA/Ionomycin and Brefeldin A/Monensin followed by intracellular IL-4 staining ( G, H ). The proportion of CD4 + IL-4 + T cells ( I ) was increased in T cells isolated from infected WT mice.

    Techniques Used: Mouse Assay, Staining, Infection, Isolation, Quantitative RT-PCR, Incubation

    33) Product Images from "C reactive protein impairs adaptive immunity in immune cells of patients with melanoma"

    Article Title: C reactive protein impairs adaptive immunity in immune cells of patients with melanoma

    Journal: Journal for Immunotherapy of Cancer

    doi: 10.1136/jitc-2019-000234

    C reactive protein (CRP) affects the function of activated T cells. Expression of checkpoint, stimulatory and cytolytic proteins by flow cytometry on activated CD8 (A) and CD4 (B) T cells treated with CRP at 10, 20 and 40 µg/mL as a heat map. Representative data from three patients. (C) CD3+ T cells isolated from baseline peripheral blood mononuclear cells (PBMC) samples of patients with melanoma prior to any treatment were cultured for 72 hours with or without CRP at doses of 10, 40 and 100 µg/mL, and subsequently stimulated for 4 hours with phorbol 12-myristate 13-acetate (PMA)/ionomycin for analysis of intracellular cytokine production (interferon (IFN)-γ, tumor necrosis factor (TNF)-α and interleukin (IL)-2) and granzyme B. Representative data from five patients. Error bars are +SEM for triplicate samples assessed. **P
    Figure Legend Snippet: C reactive protein (CRP) affects the function of activated T cells. Expression of checkpoint, stimulatory and cytolytic proteins by flow cytometry on activated CD8 (A) and CD4 (B) T cells treated with CRP at 10, 20 and 40 µg/mL as a heat map. Representative data from three patients. (C) CD3+ T cells isolated from baseline peripheral blood mononuclear cells (PBMC) samples of patients with melanoma prior to any treatment were cultured for 72 hours with or without CRP at doses of 10, 40 and 100 µg/mL, and subsequently stimulated for 4 hours with phorbol 12-myristate 13-acetate (PMA)/ionomycin for analysis of intracellular cytokine production (interferon (IFN)-γ, tumor necrosis factor (TNF)-α and interleukin (IL)-2) and granzyme B. Representative data from five patients. Error bars are +SEM for triplicate samples assessed. **P

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

    34) Product Images from "Macrophages Mediate Increased CD8 T Cell Inflammation During Weight Loss in Formerly Obese Mice"

    Article Title: Macrophages Mediate Increased CD8 T Cell Inflammation During Weight Loss in Formerly Obese Mice

    Journal: Frontiers in Endocrinology

    doi: 10.3389/fendo.2020.00257

    Switching obese mice to a normal control diet increases frequencies of pro-inflammatory cytokine producing CD8+ T cells in adipose tissue and liver. Animals were fed for 20 weeks on a high fat diet (HFD), normal control diet (NCD) or were switched after 16 weeks of a HFD to a NCD for 4 weeks. Frequency of IFN-γ+ and TNF+ (A,C) , IL-17+ and IL-22+ (B,D) CD4+ T cells (A,B) , and CD8+ T cells (C,D) within the adipose tissue. Frequency of IFN-γ+ and TNF+ (E,G) , IL-17+ and IL-22+ (F,H) CD4+ T cells (E,F) , and CD8+ T cells (G,H) within the liver. Cytokine expression of T cells was determined following PMA/Ionomycin stimulation. * p
    Figure Legend Snippet: Switching obese mice to a normal control diet increases frequencies of pro-inflammatory cytokine producing CD8+ T cells in adipose tissue and liver. Animals were fed for 20 weeks on a high fat diet (HFD), normal control diet (NCD) or were switched after 16 weeks of a HFD to a NCD for 4 weeks. Frequency of IFN-γ+ and TNF+ (A,C) , IL-17+ and IL-22+ (B,D) CD4+ T cells (A,B) , and CD8+ T cells (C,D) within the adipose tissue. Frequency of IFN-γ+ and TNF+ (E,G) , IL-17+ and IL-22+ (F,H) CD4+ T cells (E,F) , and CD8+ T cells (G,H) within the liver. Cytokine expression of T cells was determined following PMA/Ionomycin stimulation. * p

    Techniques Used: Mouse Assay, Expressing

    35) Product Images from "HSV activates Akt to trigger calcium release and promote viral entry: novel candidate target for treatment and suppression"

    Article Title: HSV activates Akt to trigger calcium release and promote viral entry: novel candidate target for treatment and suppression

    Journal: The FASEB Journal

    doi: 10.1096/fj.12-220285

    Akt is required for HSV-induced calcium release, and treatment with ionomycin partially overcomes the block to infection in Akt-silenced cells. A ) CaSki cells were loaded with Calcium Green 72 h post-transfection with the indicated siRNA and synchronously
    Figure Legend Snippet: Akt is required for HSV-induced calcium release, and treatment with ionomycin partially overcomes the block to infection in Akt-silenced cells. A ) CaSki cells were loaded with Calcium Green 72 h post-transfection with the indicated siRNA and synchronously

    Techniques Used: Blocking Assay, Infection, Transfection

    36) Product Images from "Defective IgA response to atypical intestinal commensals in IL-21 receptor deficiency reshapes immune cell homeostasis and mucosal immunity"

    Article Title: Defective IgA response to atypical intestinal commensals in IL-21 receptor deficiency reshapes immune cell homeostasis and mucosal immunity

    Journal: Mucosal immunology

    doi: 10.1038/s41385-018-0056-x

    Increased Th17 and Treg cells in the small intestine of SFB + IL-21R KO mice. Representative flow cytometry plots of various T cell populations in SILP. a , CD4 + TCRβ + cells (red square) were gated on CD45.2 + and then plotted CD4 versus Foxp3. Foxp3 - cells (green square) were further plotted CD4 versus RORγt. The graphs indicate the frequency ( n =4 or 8) and cell number ( n =4) of various T cell populations. b , CD45.2 + CD90 + TCRβ + cells were plotted IL-17 versus IL-22 following in vitro PMA and ionomycin stimulation for 4 hours (a pool of 2 mice). Isotype controls for IL-17 and IL22 are shown. IL-22 + includes both IL-22 single-positive and IL-17/IL-22 doublepositive cells (IL-17; n =4 and IL-22; n =3). Ratio paired t -test was used for comparison of cells numbers. c , Lin - (CD3e - CD11b - TCRγδ - ) NK1.1 - cells (red square) were gated on CD45.2 + and then plotted RORγt versus CD90 for RORγt + ILC3s ( n =4). Data summarize 2–4 independent experiments and are shown as mean ± SEM.
    Figure Legend Snippet: Increased Th17 and Treg cells in the small intestine of SFB + IL-21R KO mice. Representative flow cytometry plots of various T cell populations in SILP. a , CD4 + TCRβ + cells (red square) were gated on CD45.2 + and then plotted CD4 versus Foxp3. Foxp3 - cells (green square) were further plotted CD4 versus RORγt. The graphs indicate the frequency ( n =4 or 8) and cell number ( n =4) of various T cell populations. b , CD45.2 + CD90 + TCRβ + cells were plotted IL-17 versus IL-22 following in vitro PMA and ionomycin stimulation for 4 hours (a pool of 2 mice). Isotype controls for IL-17 and IL22 are shown. IL-22 + includes both IL-22 single-positive and IL-17/IL-22 doublepositive cells (IL-17; n =4 and IL-22; n =3). Ratio paired t -test was used for comparison of cells numbers. c , Lin - (CD3e - CD11b - TCRγδ - ) NK1.1 - cells (red square) were gated on CD45.2 + and then plotted RORγt versus CD90 for RORγt + ILC3s ( n =4). Data summarize 2–4 independent experiments and are shown as mean ± SEM.

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

    37) Product Images from "Interleukin (IL)-18 Binding Protein Deficiency Disrupts Natural Killer Cell Maturation and Diminishes Circulating IL-18"

    Article Title: Interleukin (IL)-18 Binding Protein Deficiency Disrupts Natural Killer Cell Maturation and Diminishes Circulating IL-18

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2017.01020

    Natural killer (NK) cells from interleukin (IL)-18BPKO mice are polarized toward greater TNF-α production and reduced IFN-γ production. NK cells were stimulated for 24 h with either IL-18 (50 ng/mL), IL-18 (50 ng/mL) + IL-2 (2 ng/mL), or indirectly with LPS (500 ng/mL) and then treated with PMA and ionomycin (PMA/Iono) for 5 h prior to intracellular labeling of IFN-γ and TNF-α. (A) Representative gating for each treatment from wild-type (WT) and IL-18BPKO mice. (B) The proportion of IFN-γ + , TNF-α − NK cells was reduced among unstimulated and stimulated NK cells. (C,D) IFN-γ + , TNF-α + , and IFN-γ − , TNF-α + NK cells from IL-18BPKO mice were increased among unstimulated and stimulated NK cells. (E) IFN-γ − , TNF-α − NK cells were elevated among IL-18BPKO mice in unstimulated and LPS stimulated groups. Results from two unique experiments from eight WT and seven IL-18BPKO mice. Bars represent median. Significance calculated with Mann–Whitney U test. * p
    Figure Legend Snippet: Natural killer (NK) cells from interleukin (IL)-18BPKO mice are polarized toward greater TNF-α production and reduced IFN-γ production. NK cells were stimulated for 24 h with either IL-18 (50 ng/mL), IL-18 (50 ng/mL) + IL-2 (2 ng/mL), or indirectly with LPS (500 ng/mL) and then treated with PMA and ionomycin (PMA/Iono) for 5 h prior to intracellular labeling of IFN-γ and TNF-α. (A) Representative gating for each treatment from wild-type (WT) and IL-18BPKO mice. (B) The proportion of IFN-γ + , TNF-α − NK cells was reduced among unstimulated and stimulated NK cells. (C,D) IFN-γ + , TNF-α + , and IFN-γ − , TNF-α + NK cells from IL-18BPKO mice were increased among unstimulated and stimulated NK cells. (E) IFN-γ − , TNF-α − NK cells were elevated among IL-18BPKO mice in unstimulated and LPS stimulated groups. Results from two unique experiments from eight WT and seven IL-18BPKO mice. Bars represent median. Significance calculated with Mann–Whitney U test. * p

    Techniques Used: Mouse Assay, Labeling, MANN-WHITNEY

    38) Product Images from "Shp1 regulates T cell homeostasis by limiting IL-4 signals"

    Article Title: Shp1 regulates T cell homeostasis by limiting IL-4 signals

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20122239

    T cells skew to Th2 in the absence of Shp1. (A) Naive (CD44 lo ) CD4 + T cells were isolated, stimulated for 3 d with α-CD3/CD28, and then restimulated with PMA/Ionomycin 4 d later. Cells were harvested and stained 6 h after restimulation. (B) Cells were stimulated and analyzed as in A. Percentages of cells staining positive for IFN γ or IL-4. Data are representative of three independent experiments and are displayed as ± standard error; n = 4. (C) Concentration of IgE and IgG2a in sera from the indicated unmanipulated age-matched mice. Horizontal bars represent sample means; n = 8. (D) T cells were stimulated with the indicated concentrations of IL-4 for 30 min and then fixed and stained for p-Stat6. Histograms are gated on CD44 lo T cells. (E) T cells were stimulated with 10 ng/ml IL-4 for 30 min and were then washed three times with media. Cells were harvested at the indicated times and analyzed as in D . Data are representative of three independent experiments. Statistical analyses of data in B and C were performed by Student’s t test; ns, P ≥ 0.05; *, P
    Figure Legend Snippet: T cells skew to Th2 in the absence of Shp1. (A) Naive (CD44 lo ) CD4 + T cells were isolated, stimulated for 3 d with α-CD3/CD28, and then restimulated with PMA/Ionomycin 4 d later. Cells were harvested and stained 6 h after restimulation. (B) Cells were stimulated and analyzed as in A. Percentages of cells staining positive for IFN γ or IL-4. Data are representative of three independent experiments and are displayed as ± standard error; n = 4. (C) Concentration of IgE and IgG2a in sera from the indicated unmanipulated age-matched mice. Horizontal bars represent sample means; n = 8. (D) T cells were stimulated with the indicated concentrations of IL-4 for 30 min and then fixed and stained for p-Stat6. Histograms are gated on CD44 lo T cells. (E) T cells were stimulated with 10 ng/ml IL-4 for 30 min and were then washed three times with media. Cells were harvested at the indicated times and analyzed as in D . Data are representative of three independent experiments. Statistical analyses of data in B and C were performed by Student’s t test; ns, P ≥ 0.05; *, P

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

    39) Product Images from "A Subset of CXCR5+CD8+ T Cells in the Germinal Centers From Human Tonsils and Lymph Nodes Help B Cells Produce Immunoglobulins"

    Article Title: A Subset of CXCR5+CD8+ T Cells in the Germinal Centers From Human Tonsils and Lymph Nodes Help B Cells Produce Immunoglobulins

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.02287

    The expression of cytokines by CXCR5 + CD8 + memory T Cells from tonsils, lymph nodes and PBMCs. The mononuclear cells from tonsils, lymph nodes and blood were cultured with or without PMA plus ionomycin in the presence of BFA for 6 h. FACS analysis of IL-21, IL-4, and IFN-γ expression in CXCR5 + and CXCR5 − CD8 + memory T Cells (A–C) . Data represent mean ± SD, and compared with two-tailed unpaired t -test (D , n = 11). * P
    Figure Legend Snippet: The expression of cytokines by CXCR5 + CD8 + memory T Cells from tonsils, lymph nodes and PBMCs. The mononuclear cells from tonsils, lymph nodes and blood were cultured with or without PMA plus ionomycin in the presence of BFA for 6 h. FACS analysis of IL-21, IL-4, and IFN-γ expression in CXCR5 + and CXCR5 − CD8 + memory T Cells (A–C) . Data represent mean ± SD, and compared with two-tailed unpaired t -test (D , n = 11). * P

    Techniques Used: Expressing, Cell Culture, FACS, Two Tailed Test

    Sorted CXCR5 + CD8 + T cells expressed higher levels of cytokines at protein and mRNA levels than CXCR5 − CD8 + T cells. Tonsil CD8 + T cells were isolated using the appropriate microbeads, and the purity of the cells was ≥98%. CXCR5 + CD8 + and CXCR5 − CD8 + T cells from tonsil CD8 + T cells were further sorted by flow cytometry (A) . Purified CD8 + , sorted CXCR5 + CD8 + and CXCR5 − CD8 + T cells were stimulated with PMA and ionomycin. The supernatants from the cultures were analyzed by ELISA for the production of IL-21, IFN-γ, and IL-4 (B) . The frequency of IL-21 and IFN-γ-producing cells was assessed by ELISPOT. The left panel shows representative counting of spot-forming cells (SFC) and the right panel shows the frequency of IL-21 and IFN-γ-producing cells as the mean with individual data points (C) . FACS analysis of IL-21, and IFN-γ expression in CD8 + , CXCR5 + CD8 + , CXCR5 − CD8 + T cells (D) . The levels of IL-21, IFN-γ and GAPDH mRNA in CD8 + , CXCR5 + CD8 + , CXCR5 − CD8 + T cells were determined by PCR (E) , and the ratio of IL-21 or IFN-γ to GAPDH were quantified by densitometry (F) . Statistical significance was compared with Mann–Whitney test. * P
    Figure Legend Snippet: Sorted CXCR5 + CD8 + T cells expressed higher levels of cytokines at protein and mRNA levels than CXCR5 − CD8 + T cells. Tonsil CD8 + T cells were isolated using the appropriate microbeads, and the purity of the cells was ≥98%. CXCR5 + CD8 + and CXCR5 − CD8 + T cells from tonsil CD8 + T cells were further sorted by flow cytometry (A) . Purified CD8 + , sorted CXCR5 + CD8 + and CXCR5 − CD8 + T cells were stimulated with PMA and ionomycin. The supernatants from the cultures were analyzed by ELISA for the production of IL-21, IFN-γ, and IL-4 (B) . The frequency of IL-21 and IFN-γ-producing cells was assessed by ELISPOT. The left panel shows representative counting of spot-forming cells (SFC) and the right panel shows the frequency of IL-21 and IFN-γ-producing cells as the mean with individual data points (C) . FACS analysis of IL-21, and IFN-γ expression in CD8 + , CXCR5 + CD8 + , CXCR5 − CD8 + T cells (D) . The levels of IL-21, IFN-γ and GAPDH mRNA in CD8 + , CXCR5 + CD8 + , CXCR5 − CD8 + T cells were determined by PCR (E) , and the ratio of IL-21 or IFN-γ to GAPDH were quantified by densitometry (F) . Statistical significance was compared with Mann–Whitney test. * P

    Techniques Used: Isolation, Flow Cytometry, Cytometry, Purification, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot, FACS, Expressing, Polymerase Chain Reaction, MANN-WHITNEY

    40) Product Images from "Trace Levels of Staphylococcal Enterotoxin Bioactivity Are Concealed in a Mucosal Niche during Pulmonary Inflammation"

    Article Title: Trace Levels of Staphylococcal Enterotoxin Bioactivity Are Concealed in a Mucosal Niche during Pulmonary Inflammation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0141548

    Lactoferrin significantly inhibits early intracellular TNF synthesis in naïve over that made by effector T cells. ( A ) Splenocytes from naïve mice were incubated with lactoferrin or BSA (5 mg/ml), in the presence of BFA and stimulated with or without PMA + Ionomycin for 4 h at 37°C. Cells were stained for CD4 and TCR Vβ3, fixed, permeabilized and stained for intracellular TNF. Representative dot plots show the gating strategy. The percentage of SEA-specific T cells: CD4 + Vβ3 + , is indicated in top right quadrant (left panels) and the percentage of those cells producing TNF is indicated as histograms in the right panels. ( B ) The mean percentage +/- standard error of the mean of CD4 + Vβ3 + cells producing TNF from 3 independent biological replicates was analyzed as described in A is shown. Hence, naïve and effector splenocytes were obtained as described in the legend of Fig 6 . One set of cells were directly incubated with lactoferrin or BSA in the presence of BFA and PMA + Ionomycin ( No wash , left panels) and stained as described in A . Another set of cells ( Washed , right panels) was first incubated for 3 h with lactoferrin or BSA, washed 4 times as described in legend of Fig 6c and then stimulated with or without PMA + Ionomycin for 4 h at 37°C. Data are combined from 3 experiments with n = 3 and displayed as mean +/- standard error of the mean. Statistical significance between BSA and lactoferrin was evaluated by two-tailed Student’s t tests. * p
    Figure Legend Snippet: Lactoferrin significantly inhibits early intracellular TNF synthesis in naïve over that made by effector T cells. ( A ) Splenocytes from naïve mice were incubated with lactoferrin or BSA (5 mg/ml), in the presence of BFA and stimulated with or without PMA + Ionomycin for 4 h at 37°C. Cells were stained for CD4 and TCR Vβ3, fixed, permeabilized and stained for intracellular TNF. Representative dot plots show the gating strategy. The percentage of SEA-specific T cells: CD4 + Vβ3 + , is indicated in top right quadrant (left panels) and the percentage of those cells producing TNF is indicated as histograms in the right panels. ( B ) The mean percentage +/- standard error of the mean of CD4 + Vβ3 + cells producing TNF from 3 independent biological replicates was analyzed as described in A is shown. Hence, naïve and effector splenocytes were obtained as described in the legend of Fig 6 . One set of cells were directly incubated with lactoferrin or BSA in the presence of BFA and PMA + Ionomycin ( No wash , left panels) and stained as described in A . Another set of cells ( Washed , right panels) was first incubated for 3 h with lactoferrin or BSA, washed 4 times as described in legend of Fig 6c and then stimulated with or without PMA + Ionomycin for 4 h at 37°C. Data are combined from 3 experiments with n = 3 and displayed as mean +/- standard error of the mean. Statistical significance between BSA and lactoferrin was evaluated by two-tailed Student’s t tests. * p

    Techniques Used: Mouse Assay, Incubation, Staining, Two Tailed Test

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    Cell Culture:

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    Article Snippet: .. )] 200 mM L-glutamine CaCl2 •2H2 O Cell culture pipettes and flasks Coverslips, glass, sterile, 25 mm (Fisher No. 1) D-glucose )] Dulbecco’s modified Eagle medium containing 25 mM glucose (DMEM) Fetal bovine serum Forskolin (Sigma-Aldrich, #F-3917) )] Hepes-sodium salt Ionomycin, Ca2+ salt (Molecular Probes, #I-24222) KCl KH2 PO4 Lipofectamine-2000 (Invitrogen) Mammalian expression vector for the biosynthetic FRET sensor Epac1-camps (can be obtained from Martin J. Lohse; see contact information above) MgCl2 •7H2 O Microcentrifuge tubes Micropipettors MIN6 mouse insulinoma cell line NaCl NaHCO3 Penicillin (10,000 units/ml)-streptomycin (10,000 μg/ml) (pen-strep) ) Tissue culture plates, 6-well .. Laminar flow hood Tissue culture incubator at 37°C, 5% CO2

    Concentration Assay:

    Article Title: Z-scan Fluorescence Profile Deconvolution of Cytosolic and Membrane-associated Protein Populations
    Article Snippet: .. Pre-warmed L-15 medium (100 μL) containing Texas Red and ionomycin calcium salt (Life Technologies, Carlsbad, CA) was later added to the well, for a final concentration of 1.26 mM for Ca2+ and 5 or 10 μM for ionomycin. ..

    Article Title: Benzodiazepinone Derivatives Protect against Endoplasmic Reticulum Stress-Mediated Cell Death in Human Neuronal Cell Lines
    Article Snippet: .. After 1 h incubation at 37 °C, 2.5 μL of inducers including thapsigargin, 2-cyano-3,12-dioxooleana-1,9-dien-28-imidazolide (CDDO-IM, Toronto Research Chemicals Inc., C228090), ionomycin (Life Technologies, I-24222), or MG132 (Sigma-Aldrich, C2211) in 2% DMSO was added to reach a corresponding concentration and 2.5 μL of 2% DMSO was used as control. .. The plates were incubated for 18 h, and the reporter signal was recorded with a BMG POLARstar Omega plate reader in luminescence mode after adding 25 μL of Steady-Glo luciferase assay reagent (Promega, E2510).

    Incubation:

    Article Title: Shared and Unique Features Distinguishing Follicular T Helper and Regulatory Cells of Peripheral Lymph Node and Peyer’s Patches
    Article Snippet: .. Total cell preparations were plated at density of 5 × 106 /ml and incubated at 37°C for 4 h in RPMI 1640/10% FCS medium supplemented with 10 µM KN-62, Ionomycin 1.5 µg/ml (Invitrogen cat# I-24222), PMA 50 ng/ml (Calbiochem cat# 524400) and Brefeldin A 10 µg/ml (Sigma-Aldrich cat# B6542). .. During this step control cells are kept on ice in FACS buffer.

    Article Title: Benzodiazepinone Derivatives Protect against Endoplasmic Reticulum Stress-Mediated Cell Death in Human Neuronal Cell Lines
    Article Snippet: .. After 1 h incubation at 37 °C, 2.5 μL of inducers including thapsigargin, 2-cyano-3,12-dioxooleana-1,9-dien-28-imidazolide (CDDO-IM, Toronto Research Chemicals Inc., C228090), ionomycin (Life Technologies, I-24222), or MG132 (Sigma-Aldrich, C2211) in 2% DMSO was added to reach a corresponding concentration and 2.5 μL of 2% DMSO was used as control. .. The plates were incubated for 18 h, and the reporter signal was recorded with a BMG POLARstar Omega plate reader in luminescence mode after adding 25 μL of Steady-Glo luciferase assay reagent (Promega, E2510).

    Expressing:

    Article Title: Simultaneous Optical Measurements of Cytosolic Ca2+ and cAMP in Single Cells
    Article Snippet: .. )] 200 mM L-glutamine CaCl2 •2H2 O Cell culture pipettes and flasks Coverslips, glass, sterile, 25 mm (Fisher No. 1) D-glucose )] Dulbecco’s modified Eagle medium containing 25 mM glucose (DMEM) Fetal bovine serum Forskolin (Sigma-Aldrich, #F-3917) )] Hepes-sodium salt Ionomycin, Ca2+ salt (Molecular Probes, #I-24222) KCl KH2 PO4 Lipofectamine-2000 (Invitrogen) Mammalian expression vector for the biosynthetic FRET sensor Epac1-camps (can be obtained from Martin J. Lohse; see contact information above) MgCl2 •7H2 O Microcentrifuge tubes Micropipettors MIN6 mouse insulinoma cell line NaCl NaHCO3 Penicillin (10,000 units/ml)-streptomycin (10,000 μg/ml) (pen-strep) ) Tissue culture plates, 6-well .. Laminar flow hood Tissue culture incubator at 37°C, 5% CO2

    Article Title: Independent and inter-dependent immunoregulatory effects of NCF1 and NOS2 in experimental autoimmune encephalomyelitis
    Article Snippet: .. To measure intracellular ROS/RNS, the staining of 3 μM Dihydrorhodamine 123 (DHR 123, ThermoFisher, Catalog No. D23806, Sweden), or 5 μM 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (DCF, ThermoFisher, Cat. No. C400, Sweden) was conducted respectively after cell surface markers staining, followed by stimulation of 100 ng/mL of phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich Co., CAS No. 16561-29-8, Sweden) alone or plus 1 μg/mL of ionomycin (ThermoFisher, Catalog No. I24222, Sweden) for 30 min. To detect the intracellular expression of cytokines, the cells were stimulated with 100 ng/mL of PMA and 1 μg/mL of ionomycin in the presence of 5 μg/mL of brefeldin A (BFA, ThermoFisher, Catalog No. B7450, Sweden) for 4 h at a humidified 37 °C, 5% CO2 incubator. .. The stock solutions of PMA, ionomycin, and BFA were prepared with dimethylsulfoxide (DMSO, Sigma-Aldrich Co., CAS No. 67-68-5, Sweden).

    Modification:

    Article Title: Simultaneous Optical Measurements of Cytosolic Ca2+ and cAMP in Single Cells
    Article Snippet: .. )] 200 mM L-glutamine CaCl2 •2H2 O Cell culture pipettes and flasks Coverslips, glass, sterile, 25 mm (Fisher No. 1) D-glucose )] Dulbecco’s modified Eagle medium containing 25 mM glucose (DMEM) Fetal bovine serum Forskolin (Sigma-Aldrich, #F-3917) )] Hepes-sodium salt Ionomycin, Ca2+ salt (Molecular Probes, #I-24222) KCl KH2 PO4 Lipofectamine-2000 (Invitrogen) Mammalian expression vector for the biosynthetic FRET sensor Epac1-camps (can be obtained from Martin J. Lohse; see contact information above) MgCl2 •7H2 O Microcentrifuge tubes Micropipettors MIN6 mouse insulinoma cell line NaCl NaHCO3 Penicillin (10,000 units/ml)-streptomycin (10,000 μg/ml) (pen-strep) ) Tissue culture plates, 6-well .. Laminar flow hood Tissue culture incubator at 37°C, 5% CO2

    Staining:

    Article Title: Independent and inter-dependent immunoregulatory effects of NCF1 and NOS2 in experimental autoimmune encephalomyelitis
    Article Snippet: .. To measure intracellular ROS/RNS, the staining of 3 μM Dihydrorhodamine 123 (DHR 123, ThermoFisher, Catalog No. D23806, Sweden), or 5 μM 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (DCF, ThermoFisher, Cat. No. C400, Sweden) was conducted respectively after cell surface markers staining, followed by stimulation of 100 ng/mL of phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich Co., CAS No. 16561-29-8, Sweden) alone or plus 1 μg/mL of ionomycin (ThermoFisher, Catalog No. I24222, Sweden) for 30 min. To detect the intracellular expression of cytokines, the cells were stimulated with 100 ng/mL of PMA and 1 μg/mL of ionomycin in the presence of 5 μg/mL of brefeldin A (BFA, ThermoFisher, Catalog No. B7450, Sweden) for 4 h at a humidified 37 °C, 5% CO2 incubator. .. The stock solutions of PMA, ionomycin, and BFA were prepared with dimethylsulfoxide (DMSO, Sigma-Aldrich Co., CAS No. 67-68-5, Sweden).

    Plasmid Preparation:

    Article Title: Simultaneous Optical Measurements of Cytosolic Ca2+ and cAMP in Single Cells
    Article Snippet: .. )] 200 mM L-glutamine CaCl2 •2H2 O Cell culture pipettes and flasks Coverslips, glass, sterile, 25 mm (Fisher No. 1) D-glucose )] Dulbecco’s modified Eagle medium containing 25 mM glucose (DMEM) Fetal bovine serum Forskolin (Sigma-Aldrich, #F-3917) )] Hepes-sodium salt Ionomycin, Ca2+ salt (Molecular Probes, #I-24222) KCl KH2 PO4 Lipofectamine-2000 (Invitrogen) Mammalian expression vector for the biosynthetic FRET sensor Epac1-camps (can be obtained from Martin J. Lohse; see contact information above) MgCl2 •7H2 O Microcentrifuge tubes Micropipettors MIN6 mouse insulinoma cell line NaCl NaHCO3 Penicillin (10,000 units/ml)-streptomycin (10,000 μg/ml) (pen-strep) ) Tissue culture plates, 6-well .. Laminar flow hood Tissue culture incubator at 37°C, 5% CO2

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    Thermo Fisher ionomycin
    Malt1 R155A knock‐in mice express a catalytically active form of Malt1 but lack self‐cleavage activity (A) Schematic representation of Malt1 protein and its functional death domain (DD), immunoglobulin‐like domains (Ig), auto processing site and catalytic site. (B) MALT1 protease reporter assays of 293T‐BM cells transiently expressing a mouse wild‐type Malt1 (mp‐Malt1), the self‐cleavage resistant R155A mutant (mp‐Malt1‐SR), the protease‐death C472A mutant (mp‐Malt1‐PD) or empty vector (mock). Malt1 protease‐dependent luciferase activity is shown as fold induction of vector‐transfected cells. The data are shown as mean +SD of four pooled independent experiments, which were each performed in triplicates. ( C ) Cell lysates were immunoblotted with the indicated antibodies to detect MALT1, its N‐ and C‐terminal auto‐cleavage fragments (p16 and p76 respectively) and its proteolytic targets. Arrows indicate the cleavage fragments generated by Malt1 protease activity. (D) Immunoblot analysis of spleen of Malt1 +/+ , Malt1 +/SR , Malt1 SR/SR and Malt1 PD/PD mice. Splenocytes from 4 mice were combined and then incubated with or without PMA and <t>ionomycin</t> for the indicated times. A non‐specific band generated with the Mcpip1 antibody was used a loading control. Data are representative of three independent experiments.
    Ionomycin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 46 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ionomycin/product/Thermo Fisher
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    Thermo Fisher pma ionomycin
    FWGP activates NK cell in immunocompetent mice. BALB/c animals were treated with FWGP (140 mg/kg) or PBS for 3 days before spleens were dissected. Splenocytes were T-cell depleted and used as effector cells in functional assays. (A) For killing assays, target cells were CFSE-labeled YAC-1. Data points represent the mean±SD of the % CFSE + FVD + cells. (B) For degranulation assays YAC-1 cells were used as target. Data points represent the median±SD fluorescence intensity (as % of max) of CD107a staining. Maximum degranulation was defined as the CD107a signal intensity in cells stimulated with <t>PMA+ionomycin</t> (n = 3; * p
    Pma Ionomycin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 32 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Malt1 R155A knock‐in mice express a catalytically active form of Malt1 but lack self‐cleavage activity (A) Schematic representation of Malt1 protein and its functional death domain (DD), immunoglobulin‐like domains (Ig), auto processing site and catalytic site. (B) MALT1 protease reporter assays of 293T‐BM cells transiently expressing a mouse wild‐type Malt1 (mp‐Malt1), the self‐cleavage resistant R155A mutant (mp‐Malt1‐SR), the protease‐death C472A mutant (mp‐Malt1‐PD) or empty vector (mock). Malt1 protease‐dependent luciferase activity is shown as fold induction of vector‐transfected cells. The data are shown as mean +SD of four pooled independent experiments, which were each performed in triplicates. ( C ) Cell lysates were immunoblotted with the indicated antibodies to detect MALT1, its N‐ and C‐terminal auto‐cleavage fragments (p16 and p76 respectively) and its proteolytic targets. Arrows indicate the cleavage fragments generated by Malt1 protease activity. (D) Immunoblot analysis of spleen of Malt1 +/+ , Malt1 +/SR , Malt1 SR/SR and Malt1 PD/PD mice. Splenocytes from 4 mice were combined and then incubated with or without PMA and ionomycin for the indicated times. A non‐specific band generated with the Mcpip1 antibody was used a loading control. Data are representative of three independent experiments.

    Journal: European Journal of Immunology

    Article Title: Malt1 self‐cleavage is critical for regulatory T cell homeostasis and anti‐tumor immunity in mice

    doi: 10.1002/eji.201847597

    Figure Lengend Snippet: Malt1 R155A knock‐in mice express a catalytically active form of Malt1 but lack self‐cleavage activity (A) Schematic representation of Malt1 protein and its functional death domain (DD), immunoglobulin‐like domains (Ig), auto processing site and catalytic site. (B) MALT1 protease reporter assays of 293T‐BM cells transiently expressing a mouse wild‐type Malt1 (mp‐Malt1), the self‐cleavage resistant R155A mutant (mp‐Malt1‐SR), the protease‐death C472A mutant (mp‐Malt1‐PD) or empty vector (mock). Malt1 protease‐dependent luciferase activity is shown as fold induction of vector‐transfected cells. The data are shown as mean +SD of four pooled independent experiments, which were each performed in triplicates. ( C ) Cell lysates were immunoblotted with the indicated antibodies to detect MALT1, its N‐ and C‐terminal auto‐cleavage fragments (p16 and p76 respectively) and its proteolytic targets. Arrows indicate the cleavage fragments generated by Malt1 protease activity. (D) Immunoblot analysis of spleen of Malt1 +/+ , Malt1 +/SR , Malt1 SR/SR and Malt1 PD/PD mice. Splenocytes from 4 mice were combined and then incubated with or without PMA and ionomycin for the indicated times. A non‐specific band generated with the Mcpip1 antibody was used a loading control. Data are representative of three independent experiments.

    Article Snippet: As positive control, cells were stimulated with 75 ng/ml PMA – 150 ng/mL ionomycin for 4 h. Primary mouse naïve CD4+ T cells or total CD4+ T cells were isolated using negative selection kits (Thermo Fisher Scientific).

    Techniques: Knock-In, Mouse Assay, Activity Assay, Functional Assay, Expressing, Mutagenesis, Plasmid Preparation, Luciferase, Transfection, Generated, Incubation

    Inulanolide A inhibits NFAT1-mediated MDM2 transcription. (A) LNCaP and PC3 cells were exposed to InuA (5 μM) for 24 h in the presence or absence of ionomycin (ION). Nuclear proteins were extracted and incubated with an MDM2 probe, followed by an EMSA assay. NE, nuclear extract. (B) LNCaP and PC3 cells were exposed to InuA (5 μM) for 24 h. The crosslinked chromatin was immunoprecipitated with anti-NFAT1 or IgG antibodies, followed by a real-time PCR analysis. (C) LNCaP and PC3 cells were transfected with NFAT1 siRNA or the respective control siRNA for 36 h, followed by exposure to InuA at the indicated concentrations for 24 h. The relative MDM2 mRNA levels and the protein levels of NFAT1 and MDM2 were determined by quantitative real-time PCR and Western blotting, respectively. (D) LNCaP and PC3 cells were co-transfected with DN-NFAT and MDM2 P2 promoter luciferase for 24 h, followed by exposure to InuA at the indicated concentrations for 24 h. The relative luciferase levels were then determined using a dual-reporter gene detection system. The protein levels of DN-NFAT and MDM2 were determined by a Western blot analysis. Data are representative of at least three experiments ( ∗ P

    Journal: Frontiers in Pharmacology

    Article Title: Targeting the NFAT1-MDM2-MDMX Network Inhibits the Proliferation and Invasion of Prostate Cancer Cells, Independent of p53 and Androgen

    doi: 10.3389/fphar.2017.00917

    Figure Lengend Snippet: Inulanolide A inhibits NFAT1-mediated MDM2 transcription. (A) LNCaP and PC3 cells were exposed to InuA (5 μM) for 24 h in the presence or absence of ionomycin (ION). Nuclear proteins were extracted and incubated with an MDM2 probe, followed by an EMSA assay. NE, nuclear extract. (B) LNCaP and PC3 cells were exposed to InuA (5 μM) for 24 h. The crosslinked chromatin was immunoprecipitated with anti-NFAT1 or IgG antibodies, followed by a real-time PCR analysis. (C) LNCaP and PC3 cells were transfected with NFAT1 siRNA or the respective control siRNA for 36 h, followed by exposure to InuA at the indicated concentrations for 24 h. The relative MDM2 mRNA levels and the protein levels of NFAT1 and MDM2 were determined by quantitative real-time PCR and Western blotting, respectively. (D) LNCaP and PC3 cells were co-transfected with DN-NFAT and MDM2 P2 promoter luciferase for 24 h, followed by exposure to InuA at the indicated concentrations for 24 h. The relative luciferase levels were then determined using a dual-reporter gene detection system. The protein levels of DN-NFAT and MDM2 were determined by a Western blot analysis. Data are representative of at least three experiments ( ∗ P

    Article Snippet: Electrophoretic Mobility Shift Assay (EMSA) and Chromatin Immunoprecipitation (ChIP) Assay Cells were treated with InuA and/or ionomycin (ION; an activator of the Ca2+ -calcineurin-NFAT signaling pathway) for 24 h. The treated cells were extracted using the NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo Fisher Scientific, Rockford, IL, United States).

    Techniques: Incubation, Immunoprecipitation, Real-time Polymerase Chain Reaction, Transfection, Western Blot, Luciferase

    Inulanolide A promotes NFAT1 protein degradation and inhibits NFAT1 nuclear translation. (A) LNCaP and PC3 cells were treated with InuA (2 μM) for 24 h, followed by exposure to a protein synthesis inhibitor, cycloheximide (CHX, 15 μg/mL). The protein expression levels of NFAT1 were detected by Western blotting at the indicated times after exposure to CHX. Graphs (on the right) show the quantification of the immunoblotting data. (B) LNCaP and PC3 cells were co-transfected with NFAT1 and ubiquitin plasmids, followed by treatment with InuA at the indicated concentrations for 24 h. Cell lysates were subjected to immunoprecipitation with an anti-NFAT1 antibody. The ubiquitinated NFAT1 was detected using an anti-ubiquitin antibody. (C) LNCaP and PC3 cells were treated with InuA (2 μM) in the presence or absence of ionomycin (ION; 4 μM) or cyclosporine A (CsA; 2 μM) for 24 h. The nuclear and cytosolic proteins were extracted and examined by Western blotting. Lamin B and α-tubulin were, respectively, used as the internal references for the nuclear and cytosolic extracts. Data are representative of three or more experiments.

    Journal: Frontiers in Pharmacology

    Article Title: Targeting the NFAT1-MDM2-MDMX Network Inhibits the Proliferation and Invasion of Prostate Cancer Cells, Independent of p53 and Androgen

    doi: 10.3389/fphar.2017.00917

    Figure Lengend Snippet: Inulanolide A promotes NFAT1 protein degradation and inhibits NFAT1 nuclear translation. (A) LNCaP and PC3 cells were treated with InuA (2 μM) for 24 h, followed by exposure to a protein synthesis inhibitor, cycloheximide (CHX, 15 μg/mL). The protein expression levels of NFAT1 were detected by Western blotting at the indicated times after exposure to CHX. Graphs (on the right) show the quantification of the immunoblotting data. (B) LNCaP and PC3 cells were co-transfected with NFAT1 and ubiquitin plasmids, followed by treatment with InuA at the indicated concentrations for 24 h. Cell lysates were subjected to immunoprecipitation with an anti-NFAT1 antibody. The ubiquitinated NFAT1 was detected using an anti-ubiquitin antibody. (C) LNCaP and PC3 cells were treated with InuA (2 μM) in the presence or absence of ionomycin (ION; 4 μM) or cyclosporine A (CsA; 2 μM) for 24 h. The nuclear and cytosolic proteins were extracted and examined by Western blotting. Lamin B and α-tubulin were, respectively, used as the internal references for the nuclear and cytosolic extracts. Data are representative of three or more experiments.

    Article Snippet: Electrophoretic Mobility Shift Assay (EMSA) and Chromatin Immunoprecipitation (ChIP) Assay Cells were treated with InuA and/or ionomycin (ION; an activator of the Ca2+ -calcineurin-NFAT signaling pathway) for 24 h. The treated cells were extracted using the NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo Fisher Scientific, Rockford, IL, United States).

    Techniques: Expressing, Western Blot, Transfection, Immunoprecipitation

    Lower frequency of cytokine-producing antigen-presenting cells (APCs) in cannabis-using individuals as compared to noncannabis users. Multiparameter flow cytometry was used to identify the frequency of APCs within total peripheral blood mononuclear cells of cannabis-using and nonusing individuals that expressed interleukin 6 (IL-6), interleukin 10 (IL-10), interleukin 23 (IL-23), and tumor necrosis factor alpha (TNF-α) after overnight culture in the presence of phorbol myistate acetate, ionomycin, and brefeldin A . Cells were identified by first excluding doublets using forward and side scatter properties, gating on total leukocytes as determined by CD45 expression and removing dead cells with an Aqua Live/Dead viability dye. Total APCs were then identified by gating on CD3 – CD20 – HLA-DR + cells. Expression of the indicated cytokine was then determined within this population. Pooled data is accompanied by a representative flow plot showing gating for the indicated cytokine. In all plots, individuals are classified as noncannabis users or cannabis users stratified by moderate or heavy cannabis use as determined by plasma quantities of 11-nor-carboxy-tetrahydrocannabinol. Each individual is represented by a single point. Horizontal bars indicate the median value. The statistical significance of differences between each of the cannabis-using groups and the noncannabis users was determined using the Mann-Whitney test. Abbreviations: SSC, side scatter; SSChi, side scatter high.

    Journal: Clinical Infectious Diseases: An Official Publication of the Infectious Diseases Society of America

    Article Title: Heavy Cannabis Use Associated With Reduction in Activated and Inflammatory Immune Cell Frequencies in Antiretroviral Therapy–Treated Human Immunodeficiency Virus–Infected Individuals

    doi: 10.1093/cid/cix1116

    Figure Lengend Snippet: Lower frequency of cytokine-producing antigen-presenting cells (APCs) in cannabis-using individuals as compared to noncannabis users. Multiparameter flow cytometry was used to identify the frequency of APCs within total peripheral blood mononuclear cells of cannabis-using and nonusing individuals that expressed interleukin 6 (IL-6), interleukin 10 (IL-10), interleukin 23 (IL-23), and tumor necrosis factor alpha (TNF-α) after overnight culture in the presence of phorbol myistate acetate, ionomycin, and brefeldin A . Cells were identified by first excluding doublets using forward and side scatter properties, gating on total leukocytes as determined by CD45 expression and removing dead cells with an Aqua Live/Dead viability dye. Total APCs were then identified by gating on CD3 – CD20 – HLA-DR + cells. Expression of the indicated cytokine was then determined within this population. Pooled data is accompanied by a representative flow plot showing gating for the indicated cytokine. In all plots, individuals are classified as noncannabis users or cannabis users stratified by moderate or heavy cannabis use as determined by plasma quantities of 11-nor-carboxy-tetrahydrocannabinol. Each individual is represented by a single point. Horizontal bars indicate the median value. The statistical significance of differences between each of the cannabis-using groups and the noncannabis users was determined using the Mann-Whitney test. Abbreviations: SSC, side scatter; SSChi, side scatter high.

    Article Snippet: For intracellular cytokine staining, cells were stimulated with phorbol myristate acetate (PMA; 5 ng/mL; Sigma-Aldrich, St Louis, Missouri) and ionomycin (1 μM/mL; Thermo Fisher Scientific) for 10–14 hours, in the presence of brefeldin A (1 μg/mL; Sigma-Aldrich).

    Techniques: Flow Cytometry, Cytometry, Expressing, MANN-WHITNEY

    FWGP activates NK cell in immunocompetent mice. BALB/c animals were treated with FWGP (140 mg/kg) or PBS for 3 days before spleens were dissected. Splenocytes were T-cell depleted and used as effector cells in functional assays. (A) For killing assays, target cells were CFSE-labeled YAC-1. Data points represent the mean±SD of the % CFSE + FVD + cells. (B) For degranulation assays YAC-1 cells were used as target. Data points represent the median±SD fluorescence intensity (as % of max) of CD107a staining. Maximum degranulation was defined as the CD107a signal intensity in cells stimulated with PMA+ionomycin (n = 3; * p

    Journal: PLoS ONE

    Article Title: A purified, fermented, extract of Triticum aestivum has lymphomacidal activity mediated via natural killer cell activation

    doi: 10.1371/journal.pone.0190860

    Figure Lengend Snippet: FWGP activates NK cell in immunocompetent mice. BALB/c animals were treated with FWGP (140 mg/kg) or PBS for 3 days before spleens were dissected. Splenocytes were T-cell depleted and used as effector cells in functional assays. (A) For killing assays, target cells were CFSE-labeled YAC-1. Data points represent the mean±SD of the % CFSE + FVD + cells. (B) For degranulation assays YAC-1 cells were used as target. Data points represent the median±SD fluorescence intensity (as % of max) of CD107a staining. Maximum degranulation was defined as the CD107a signal intensity in cells stimulated with PMA+ionomycin (n = 3; * p

    Article Snippet: Controls included effector-only cells (non-stimulated) and cells stimulated with PMA+ionomycin (Cell Stimulation Cocktail, Thermo Fisher).

    Techniques: Mouse Assay, Functional Assay, Labeling, Fluorescence, Staining