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    Structured Review

    Thermo Fisher ionomycin
    Hyperactive DCs drive TH1-skewed immune responses, with no evidence of TH2 immunity. (A-C) WT mice were injected subcutaneously (s.c.) on the right flank with PBS or LPS or PGPC or alum alone, or with LPS plus PGPC. Alternatively, mice were injected s.c. with Alum alone or LPS plus Alum. 24 hours post immunization, the skin draining lymph nodes (dLN) were isolated. (A) The absolute number of CD11c + live cells was measured by flow cytometry. (B) CD11c + cells were isolated as CD11c + MHC - II+CD11b - then cultured onto a plate coated with agonistic anti-CD40 antibody for 24 hours. (B) IL-12p70 and (C) IL-10 secretion from dLN DCs were measured by ELISA. Means and SD of a triplicate is shown, and data are representative of at least 3 independent experiments. (D) CD40 expression by dLN DCs was measured by flow cytometry. Means and SDs of five mice are shown and are representative of 3 independent experiments. (E) WT mice were injected s.c. on the right flank with endofit-OVA protein either alone or with LPS, or with LPS plus PGPC emulsified in either incomplete Freud’s adjuvant (IFA) or in Alum as indicated. 40 days post immunization, the dLN were isolated. CD4 + were sorted then co-cultured with BMDC loaded (or not) with OVA. IFNγ, IL-10, IL-4 secretion was measured by ELISA. Means and SDs of four mice are shown and are representative of 3 independent experiments. (F) Bone marrow derived DCs ( BMDCs) pretreated with indicated stimuli were loaded with OVA protein for 1h, then incubated for 4 days with OT-II naïve CD4+ T cells. 4 days later, T cells were stimulated for 5h with PMA plus <t>ionomycin</t> in the presence of brefeldin-A and monensin. The frequency of TH1 cells as TNFa + IFNg + , and TH2 cells as IL-4 + IL-10 + were measured by intracellular staining. Data are represented as the ratio of TH1/TH2 cells. Means and SDs of five mice are shown.
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    Images

    1) Product Images from "Correction of age-associated defects in dendritic cell functions enables CD4+ T cells to eradicate tumors in the elderly"

    Article Title: Correction of age-associated defects in dendritic cell functions enables CD4+ T cells to eradicate tumors in the elderly

    Journal: bioRxiv

    doi: 10.1101/2022.11.08.515695

    Hyperactive DCs drive TH1-skewed immune responses, with no evidence of TH2 immunity. (A-C) WT mice were injected subcutaneously (s.c.) on the right flank with PBS or LPS or PGPC or alum alone, or with LPS plus PGPC. Alternatively, mice were injected s.c. with Alum alone or LPS plus Alum. 24 hours post immunization, the skin draining lymph nodes (dLN) were isolated. (A) The absolute number of CD11c + live cells was measured by flow cytometry. (B) CD11c + cells were isolated as CD11c + MHC - II+CD11b - then cultured onto a plate coated with agonistic anti-CD40 antibody for 24 hours. (B) IL-12p70 and (C) IL-10 secretion from dLN DCs were measured by ELISA. Means and SD of a triplicate is shown, and data are representative of at least 3 independent experiments. (D) CD40 expression by dLN DCs was measured by flow cytometry. Means and SDs of five mice are shown and are representative of 3 independent experiments. (E) WT mice were injected s.c. on the right flank with endofit-OVA protein either alone or with LPS, or with LPS plus PGPC emulsified in either incomplete Freud’s adjuvant (IFA) or in Alum as indicated. 40 days post immunization, the dLN were isolated. CD4 + were sorted then co-cultured with BMDC loaded (or not) with OVA. IFNγ, IL-10, IL-4 secretion was measured by ELISA. Means and SDs of four mice are shown and are representative of 3 independent experiments. (F) Bone marrow derived DCs ( BMDCs) pretreated with indicated stimuli were loaded with OVA protein for 1h, then incubated for 4 days with OT-II naïve CD4+ T cells. 4 days later, T cells were stimulated for 5h with PMA plus ionomycin in the presence of brefeldin-A and monensin. The frequency of TH1 cells as TNFa + IFNg + , and TH2 cells as IL-4 + IL-10 + were measured by intracellular staining. Data are represented as the ratio of TH1/TH2 cells. Means and SDs of five mice are shown.
    Figure Legend Snippet: Hyperactive DCs drive TH1-skewed immune responses, with no evidence of TH2 immunity. (A-C) WT mice were injected subcutaneously (s.c.) on the right flank with PBS or LPS or PGPC or alum alone, or with LPS plus PGPC. Alternatively, mice were injected s.c. with Alum alone or LPS plus Alum. 24 hours post immunization, the skin draining lymph nodes (dLN) were isolated. (A) The absolute number of CD11c + live cells was measured by flow cytometry. (B) CD11c + cells were isolated as CD11c + MHC - II+CD11b - then cultured onto a plate coated with agonistic anti-CD40 antibody for 24 hours. (B) IL-12p70 and (C) IL-10 secretion from dLN DCs were measured by ELISA. Means and SD of a triplicate is shown, and data are representative of at least 3 independent experiments. (D) CD40 expression by dLN DCs was measured by flow cytometry. Means and SDs of five mice are shown and are representative of 3 independent experiments. (E) WT mice were injected s.c. on the right flank with endofit-OVA protein either alone or with LPS, or with LPS plus PGPC emulsified in either incomplete Freud’s adjuvant (IFA) or in Alum as indicated. 40 days post immunization, the dLN were isolated. CD4 + were sorted then co-cultured with BMDC loaded (or not) with OVA. IFNγ, IL-10, IL-4 secretion was measured by ELISA. Means and SDs of four mice are shown and are representative of 3 independent experiments. (F) Bone marrow derived DCs ( BMDCs) pretreated with indicated stimuli were loaded with OVA protein for 1h, then incubated for 4 days with OT-II naïve CD4+ T cells. 4 days later, T cells were stimulated for 5h with PMA plus ionomycin in the presence of brefeldin-A and monensin. The frequency of TH1 cells as TNFa + IFNg + , and TH2 cells as IL-4 + IL-10 + were measured by intracellular staining. Data are represented as the ratio of TH1/TH2 cells. Means and SDs of five mice are shown.

    Techniques Used: Mouse Assay, Injection, Isolation, Flow Cytometry, Cell Culture, Enzyme-linked Immunosorbent Assay, Expressing, Immunofluorescence, Derivative Assay, Incubation, Staining

    Hyperactive DCs drive TH1-skewed immune responses, with no evidence of TH2 immunity. (A-C) BMDCs pretreated with indicated stimuli were loaded (or not) with OVA protein for 1h, then incubated for 4 days with OT-II naïve CD4+ T cells. (A) Supernatants were collected and IFNγ, IL-10, TNFa and IL-13 cytokine release was measured by ELISA. (B-C) T cells were stimulated for 5h with PMA plus ionomycin in the presence of brefeldin-A and monensin. (B) Representative plots of the frequency of TH1 cells as TNFa + IFNg + , and TH2 cells as IL-4 + IL-10 + and (C) The frequency of GATA3 + IL-4 + TH2 cells were measured following intracellular staining by flow cytometry. Histograms represent the frequency of IL-10 + GATA3 + . Means and SDs of five mice are shown.
    Figure Legend Snippet: Hyperactive DCs drive TH1-skewed immune responses, with no evidence of TH2 immunity. (A-C) BMDCs pretreated with indicated stimuli were loaded (or not) with OVA protein for 1h, then incubated for 4 days with OT-II naïve CD4+ T cells. (A) Supernatants were collected and IFNγ, IL-10, TNFa and IL-13 cytokine release was measured by ELISA. (B-C) T cells were stimulated for 5h with PMA plus ionomycin in the presence of brefeldin-A and monensin. (B) Representative plots of the frequency of TH1 cells as TNFa + IFNg + , and TH2 cells as IL-4 + IL-10 + and (C) The frequency of GATA3 + IL-4 + TH2 cells were measured following intracellular staining by flow cytometry. Histograms represent the frequency of IL-10 + GATA3 + . Means and SDs of five mice are shown.

    Techniques Used: Incubation, Enzyme-linked Immunosorbent Assay, Staining, Flow Cytometry, Mouse Assay

    CD4 + T cells mediate anti-tumor immunity in young and old mice (A-D) 8 weeks or 68 weeks old C57BL/6J mice were injected subcutaneously (s.c.) on the right flank with B16OVA cells. When tumors reached 2-3 mm of size, mice were either (A) left untreated (unimmunized) or (B) treated i.p. with anti-PD1 antibodies alone, (A) The percentage of mice survival was monitored every 2 days (n=10 mice per group). (B) Tumor size in mm 2 was measured every two-three days (n=3 mice per group). (C) B16OVA tumor-bearing mice were immunized with B16OVA whole tumor lysate (WTL) and LPS plus PGPC emulsified in IFA. Tumor size in mm 2 was measured every two-three days (n=5 mice per group). (D) 68 weeks old C57BL/6J mice were injected s.c. on the right flank with B16OVA cells. When tumors reached 2 mm, mice were immunized on the left flank with PBS alone or with and LPS, or with LPS plus PGPC all emulsified in IFA, or mice were immunized with LPS plus Alum. Tumor size in mm 2 was measured every two-three days (Left panel). The percentage of mice survival was monitored every 2 days (Right panel). (E) 8 weeks old or (F) 68 weeks old C57BL/6J mice were injected s.c. on the right flank with B16OVA cells. When tumors reached 2mm in size, mice were either left unimmunized or were immunized with LPS plus PGPC with or without neutralizing anti-CD4, or anti-IL-1β or anti-CD8a antibodies. Percentage of mice survival was monitored every 2 days (n=5-10 mice per group). (G) 68 weeks old mice were injected s.c. on the right flank with B16OVA cells. When tumors reached 2-3mm in size, mice were either left unimmunized or were immunized with LPS plus PGPC. 15 days post immunization, CD45 TILs were enriched using anti-CD45 microbeads. The percentage of Tregs as CD4 + Foxp3 + T cells was measured by flow cytometry (Upper panel). CD45 TILs were activated for 24 hours with anti-CD3/CD28, then treated with PMA and ionomycin for 5 hours. The ratio of IFNg over IL-10 producing CD4 + T cells in the tumor microenvironment was measured by intracellular staining. (H) 68 weeks old wild type or Ccr7 -/- or Casp1/11 -/- or Nlrp3 -/- mice were injected s.c. on the right flank with B16OVA cells. When tumors reached 2-3mm in size, mice were either left unimmunized or were immunized with WTL and LPS plus PGPC. Percentage of mice survival was monitored every 2 days (n=5 mice per group).
    Figure Legend Snippet: CD4 + T cells mediate anti-tumor immunity in young and old mice (A-D) 8 weeks or 68 weeks old C57BL/6J mice were injected subcutaneously (s.c.) on the right flank with B16OVA cells. When tumors reached 2-3 mm of size, mice were either (A) left untreated (unimmunized) or (B) treated i.p. with anti-PD1 antibodies alone, (A) The percentage of mice survival was monitored every 2 days (n=10 mice per group). (B) Tumor size in mm 2 was measured every two-three days (n=3 mice per group). (C) B16OVA tumor-bearing mice were immunized with B16OVA whole tumor lysate (WTL) and LPS plus PGPC emulsified in IFA. Tumor size in mm 2 was measured every two-three days (n=5 mice per group). (D) 68 weeks old C57BL/6J mice were injected s.c. on the right flank with B16OVA cells. When tumors reached 2 mm, mice were immunized on the left flank with PBS alone or with and LPS, or with LPS plus PGPC all emulsified in IFA, or mice were immunized with LPS plus Alum. Tumor size in mm 2 was measured every two-three days (Left panel). The percentage of mice survival was monitored every 2 days (Right panel). (E) 8 weeks old or (F) 68 weeks old C57BL/6J mice were injected s.c. on the right flank with B16OVA cells. When tumors reached 2mm in size, mice were either left unimmunized or were immunized with LPS plus PGPC with or without neutralizing anti-CD4, or anti-IL-1β or anti-CD8a antibodies. Percentage of mice survival was monitored every 2 days (n=5-10 mice per group). (G) 68 weeks old mice were injected s.c. on the right flank with B16OVA cells. When tumors reached 2-3mm in size, mice were either left unimmunized or were immunized with LPS plus PGPC. 15 days post immunization, CD45 TILs were enriched using anti-CD45 microbeads. The percentage of Tregs as CD4 + Foxp3 + T cells was measured by flow cytometry (Upper panel). CD45 TILs were activated for 24 hours with anti-CD3/CD28, then treated with PMA and ionomycin for 5 hours. The ratio of IFNg over IL-10 producing CD4 + T cells in the tumor microenvironment was measured by intracellular staining. (H) 68 weeks old wild type or Ccr7 -/- or Casp1/11 -/- or Nlrp3 -/- mice were injected s.c. on the right flank with B16OVA cells. When tumors reached 2-3mm in size, mice were either left unimmunized or were immunized with WTL and LPS plus PGPC. Percentage of mice survival was monitored every 2 days (n=5 mice per group).

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

    2) Product Images from "Structural basis for the activation of the lipid scramblase TMEM16F"

    Article Title: Structural basis for the activation of the lipid scramblase TMEM16F

    Journal: Nature Communications

    doi: 10.1038/s41467-022-34497-x

    Functional relevance of residues during activation. a Conformation of α3 around the hinge residue Gly 473 (circle) in structures obtained for the mutant F518H. b Lipid transport activity of Gly 473 mutants quantified in a cellular scrambling assay. Data shows fluorescence levels normalized to WT, at elevated Ca 2+ , 600 s after application of ionomycin. Dashed line indicates mean value of WT. Bars show mean of six biological replicates (depicted as spheres), errors are s.e.m. c Current magnitudes of HEK293T cells expressing WT or G473P recorded in excised patches. Bars represent mean of individual experiments ( n = 8), errors are s.e.m. d Surface expression of G473P. Anti-myc Western blot corresponds to biotinylated constructs pulled-down from HEK293T cells expressing myc-tagged WT or G473P after surface biotinylation. Samples are derived from the same experiment, gels and blots were processed in parallel. e Interactions between residues on α-helices 4 and 6 that are in contact in the active but not the inactive state of TMEM16F. f Coupling energies between residues depicted in e . Inset shows scheme of the double-mutant cycle analysis. WT/F518A/Q623A and WT/F518A/W619A refer to the cycle quantifying the effect of mutations F518A and either Q623A or W619A relative to WT, F518H/F518A/F518H_Q623A to the cycle of F518A and F518H_Q623A relative to the mutant F518H. g Contact region between α-helices 4 and 6 in the activated state of the double-mutant F518A_Q623A Ca . Blow up shows residues that are in contact as space-filling models. h Comparison of the open conformation of the active subunits of N562A Ca and F518A_Q623A Ca . Pore-lining helices are shown as Cα-trace.
    Figure Legend Snippet: Functional relevance of residues during activation. a Conformation of α3 around the hinge residue Gly 473 (circle) in structures obtained for the mutant F518H. b Lipid transport activity of Gly 473 mutants quantified in a cellular scrambling assay. Data shows fluorescence levels normalized to WT, at elevated Ca 2+ , 600 s after application of ionomycin. Dashed line indicates mean value of WT. Bars show mean of six biological replicates (depicted as spheres), errors are s.e.m. c Current magnitudes of HEK293T cells expressing WT or G473P recorded in excised patches. Bars represent mean of individual experiments ( n = 8), errors are s.e.m. d Surface expression of G473P. Anti-myc Western blot corresponds to biotinylated constructs pulled-down from HEK293T cells expressing myc-tagged WT or G473P after surface biotinylation. Samples are derived from the same experiment, gels and blots were processed in parallel. e Interactions between residues on α-helices 4 and 6 that are in contact in the active but not the inactive state of TMEM16F. f Coupling energies between residues depicted in e . Inset shows scheme of the double-mutant cycle analysis. WT/F518A/Q623A and WT/F518A/W619A refer to the cycle quantifying the effect of mutations F518A and either Q623A or W619A relative to WT, F518H/F518A/F518H_Q623A to the cycle of F518A and F518H_Q623A relative to the mutant F518H. g Contact region between α-helices 4 and 6 in the activated state of the double-mutant F518A_Q623A Ca . Blow up shows residues that are in contact as space-filling models. h Comparison of the open conformation of the active subunits of N562A Ca and F518A_Q623A Ca . Pore-lining helices are shown as Cα-trace.

    Techniques Used: Functional Assay, Activation Assay, Mutagenesis, Activity Assay, Fluorescence, Expressing, Western Blot, Construct, Derivative Assay

    Characterization of activating mutants. a Ca 2+ -concentration-response relationships of mutants of Phe 518 measured in inside-out patches. b EC 50 values of Ca 2+ plotted against the normalized hydrophobicity (Eisenberg and Weiss Scale) of the respective Phe 518 mutations. Line shows a fit to the data. c Ca 2+ -concentration-response relationships of mutants of Asn 562 and d Tyr 563 measured in inside-out patches. a , c , d Data show averages of multiple experiments derived from independent cells (WT n = 10, F518H n = 5, F518Q n = 4, F518Y n = 5-6, F518A n = 5-9, F518I n = 7, N562A, n = 5, N562L n = 4, Y563A n = 5, Y563H n = 3–6), errors are s.e.m., lines show fit to a Hill equation. e , f Lipid transport of Phe 518 mutants quantified in a cellular scrambling assay. e Initial values recorded at resting Ca 2+ concentration and f levels measured 600 s after application of ionomycin, which increases intracellular Ca 2+ . Individual experiments are depicted as spheres (mock n = 10, F518I, F518A, F518Y n = 3, F518Q n = 4, F518H n = 6), errors are s.e.m. g , h Liposome-based in vitro scrambling assay of the reconstituted mutant F518H in comparison to WT. g Time-dependent fluorescence decrease upon addition of the reducing agent dithionite (t = 60 s) at 0 and 1000 µM Ca 2+ compared to WT. Data show mean of three technical replicates, errors (s.e.m.) are smaller than the displayed line width. h Ca 2+ -concentration response relationship of scrambling of the mutant F518H compared to WT obtained from three technical replicates (displayed in Supp. Fig. 2d ). Values were obtained as described in the methods. Solid line shows fit to a Hill equation, errors are s.e.m. Source data are provided as a Source Data file.
    Figure Legend Snippet: Characterization of activating mutants. a Ca 2+ -concentration-response relationships of mutants of Phe 518 measured in inside-out patches. b EC 50 values of Ca 2+ plotted against the normalized hydrophobicity (Eisenberg and Weiss Scale) of the respective Phe 518 mutations. Line shows a fit to the data. c Ca 2+ -concentration-response relationships of mutants of Asn 562 and d Tyr 563 measured in inside-out patches. a , c , d Data show averages of multiple experiments derived from independent cells (WT n = 10, F518H n = 5, F518Q n = 4, F518Y n = 5-6, F518A n = 5-9, F518I n = 7, N562A, n = 5, N562L n = 4, Y563A n = 5, Y563H n = 3–6), errors are s.e.m., lines show fit to a Hill equation. e , f Lipid transport of Phe 518 mutants quantified in a cellular scrambling assay. e Initial values recorded at resting Ca 2+ concentration and f levels measured 600 s after application of ionomycin, which increases intracellular Ca 2+ . Individual experiments are depicted as spheres (mock n = 10, F518I, F518A, F518Y n = 3, F518Q n = 4, F518H n = 6), errors are s.e.m. g , h Liposome-based in vitro scrambling assay of the reconstituted mutant F518H in comparison to WT. g Time-dependent fluorescence decrease upon addition of the reducing agent dithionite (t = 60 s) at 0 and 1000 µM Ca 2+ compared to WT. Data show mean of three technical replicates, errors (s.e.m.) are smaller than the displayed line width. h Ca 2+ -concentration response relationship of scrambling of the mutant F518H compared to WT obtained from three technical replicates (displayed in Supp. Fig. 2d ). Values were obtained as described in the methods. Solid line shows fit to a Hill equation, errors are s.e.m. Source data are provided as a Source Data file.

    Techniques Used: Concentration Assay, Derivative Assay, In Vitro, Mutagenesis, Fluorescence

    3) Product Images from "Disturbed natural killer cell homeostasis in the salivary gland enhances autoimmune pathology via IFN-γ in a mouse model of primary Sjögren’s syndrome"

    Article Title: Disturbed natural killer cell homeostasis in the salivary gland enhances autoimmune pathology via IFN-γ in a mouse model of primary Sjögren’s syndrome

    Journal: Frontiers in Medicine

    doi: 10.3389/fmed.2022.1036787

    IFN-γ production in T and NK cells in SG. CD45.2 + cells were purified from the SG tissues of pSS model mice and the purified cells were stimulated with PMA/ionomycin for 24 h. Intracellular IFN-γ or TNF-α expression was evaluated by flow cytometric analysis. (A) Intracellular IFN-γ expression in CD45.2 + , CD4 + , CD8 + T cells, and NKp46 + NK cells was analyzed. The proportions of IFN-γ + cells were evaluated, and data are presented as the means ± SD with four mice from each group. * P
    Figure Legend Snippet: IFN-γ production in T and NK cells in SG. CD45.2 + cells were purified from the SG tissues of pSS model mice and the purified cells were stimulated with PMA/ionomycin for 24 h. Intracellular IFN-γ or TNF-α expression was evaluated by flow cytometric analysis. (A) Intracellular IFN-γ expression in CD45.2 + , CD4 + , CD8 + T cells, and NKp46 + NK cells was analyzed. The proportions of IFN-γ + cells were evaluated, and data are presented as the means ± SD with four mice from each group. * P

    Techniques Used: Purification, Mouse Assay, Expressing

    4) Product Images from "Fully murine CD105-targeted CAR-T cells provide an immunocompetent model for CAR-T cell biology"

    Article Title: Fully murine CD105-targeted CAR-T cells provide an immunocompetent model for CAR-T cell biology

    Journal: Oncoimmunology

    doi: 10.1080/2162402X.2022.2131229

    mCD105-targeted CARs are functional despite the expression of inhibitory receptors. (a) Surface exhaustion markers of murine CAR-T cells (b) TCF-1/TOX expression of murine CAR-T cells (c) Ki-67 expression of murine CAR-T cells (d) Cytokine production of murine CAR-T cells after restimulation via TCR, murine CAR or PMA Ionomycin (e) murine CAR surface expression of murine CAR-T cells (f) In vitro expression of surface murine CAR after overnight stimulation through the CAR versus the TCR. All experiments were repeated with 3 different donors. All TIL analysis (a-e) was performed 14 days after CTX murine CAR infusion in a design exactly like 4A. For the TIL analysis, the data from the 3 donors were pooled. Statistics are Wilcoxon matched-pairs signed rank test or paired t-test depending on the data distribution. * p
    Figure Legend Snippet: mCD105-targeted CARs are functional despite the expression of inhibitory receptors. (a) Surface exhaustion markers of murine CAR-T cells (b) TCF-1/TOX expression of murine CAR-T cells (c) Ki-67 expression of murine CAR-T cells (d) Cytokine production of murine CAR-T cells after restimulation via TCR, murine CAR or PMA Ionomycin (e) murine CAR surface expression of murine CAR-T cells (f) In vitro expression of surface murine CAR after overnight stimulation through the CAR versus the TCR. All experiments were repeated with 3 different donors. All TIL analysis (a-e) was performed 14 days after CTX murine CAR infusion in a design exactly like 4A. For the TIL analysis, the data from the 3 donors were pooled. Statistics are Wilcoxon matched-pairs signed rank test or paired t-test depending on the data distribution. * p

    Techniques Used: Functional Assay, Expressing, In Vitro

    5) Product Images from "Monitoring correlates of SARS-CoV-2 infection in cell culture using two-photon microscopy and a novel fluorescent calcium-sensitive dye"

    Article Title: Monitoring correlates of SARS-CoV-2 infection in cell culture using two-photon microscopy and a novel fluorescent calcium-sensitive dye

    Journal: bioRxiv

    doi: 10.1101/2022.09.12.506773

    Results of the flow cytometry measurement. Dotted black line: untreated cells; Solid black line: BEEF-CP treated cells; solid orange line: ionomycin + BEEF-CP treated cells; solid red line: EGTA + BEEF-CP treated cells.
    Figure Legend Snippet: Results of the flow cytometry measurement. Dotted black line: untreated cells; Solid black line: BEEF-CP treated cells; solid orange line: ionomycin + BEEF-CP treated cells; solid red line: EGTA + BEEF-CP treated cells.

    Techniques Used: Flow Cytometry

    6) Product Images from "Peptides derived from hookworm anti-inflammatory proteins suppress inducible colitis in mice and inflammatory cytokine production by human cells"

    Article Title: Peptides derived from hookworm anti-inflammatory proteins suppress inducible colitis in mice and inflammatory cytokine production by human cells

    Journal: Frontiers in Medicine

    doi: 10.3389/fmed.2022.934852

    ES2-10 shows suppressive activity on human cytokine secretion. ES2-10 (100 μg/mL) was added to 1 × 10 6 PBMC from four genetically unrelated donors and stimulated with (A) 50 ng/mL PMA and 1 μg/mL ionomycin or (B) 10 ng/mL LPS. After 24 h incubation, cytokines from culture supernatants were quantified by CBA. (C) ES2-10 dose-response (0.1–100 μg/mL) against LPS-stimulated TNF. All results were performed in triplicate. * P
    Figure Legend Snippet: ES2-10 shows suppressive activity on human cytokine secretion. ES2-10 (100 μg/mL) was added to 1 × 10 6 PBMC from four genetically unrelated donors and stimulated with (A) 50 ng/mL PMA and 1 μg/mL ionomycin or (B) 10 ng/mL LPS. After 24 h incubation, cytokines from culture supernatants were quantified by CBA. (C) ES2-10 dose-response (0.1–100 μg/mL) against LPS-stimulated TNF. All results were performed in triplicate. * P

    Techniques Used: Activity Assay, Incubation, Crocin Bleaching Assay

    7) Product Images from "Gain-of-Function Dynamin-2 Mutations Linked to Centronuclear Myopathy Impair Ca2+-Induced Exocytosis in Human Myoblasts"

    Article Title: Gain-of-Function Dynamin-2 Mutations Linked to Centronuclear Myopathy Impair Ca2+-Induced Exocytosis in Human Myoblasts

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms231810363

    Kinetic parameters of spontaneous and ionomycin-induced pHluorin signals in C25 myoblasts. ( A ): An example of a trace of a transient fusion event induced in C25 myoblasts stimulated with 10 μM ionomycin ( top ) and its corresponding kymograph ( bottom ). The parameters analyzed were: (1) duration, (2) dwell time and (3) decay time. ( B ): A summary of these three parameters (( i ), ( ii ) and ( iii ), respectively), obtained from pHluorin signals induced in non-stimulated (N-S; 10 cells) or stimulated C25 myoblasts with 1, 5, or 10 μM ionomycin (12, 10, and 10 cells, respectively), are represented. Dots represent values from individual cells from at least three independent cultures. Horizontal lines indicate means with SEM. N. *** p
    Figure Legend Snippet: Kinetic parameters of spontaneous and ionomycin-induced pHluorin signals in C25 myoblasts. ( A ): An example of a trace of a transient fusion event induced in C25 myoblasts stimulated with 10 μM ionomycin ( top ) and its corresponding kymograph ( bottom ). The parameters analyzed were: (1) duration, (2) dwell time and (3) decay time. ( B ): A summary of these three parameters (( i ), ( ii ) and ( iii ), respectively), obtained from pHluorin signals induced in non-stimulated (N-S; 10 cells) or stimulated C25 myoblasts with 1, 5, or 10 μM ionomycin (12, 10, and 10 cells, respectively), are represented. Dots represent values from individual cells from at least three independent cultures. Horizontal lines indicate means with SEM. N. *** p

    Techniques Used:

    pHluorin signals in C25 myoblasts overexpressing WT dynamin-2 or the p.A618T or p.S619L mutations. C25 myoblasts were transfected with IRAP-pHluorin together with alternatively WT, p.A618T or p.S619L dynamin-2-mCherry plasmids. Exocytosis induced with 10 μM ionomycin was monitored 48 h later by TIRFM. ( A ): Confocal images of cells expressing IRAP-pHluorin (green) together with WT, p.A618T or p.S619L dynamin-2 (red). Scale bar = 20 μm. ( B ): Frequency of events in C25 myoblasts overexpressing WT (black; 10 cells), p.A618T (blue; 12 cells) or p.S619L (red; 14 cells) dynamin-2 in non-stimulated conditions (empty dots) or stimulated with 10 µM ionomycin (filled dots). Each dot represents the average frequency of events for individual cells from at least three independent cultures. Horizontal lines indicate means with SEM. *** p
    Figure Legend Snippet: pHluorin signals in C25 myoblasts overexpressing WT dynamin-2 or the p.A618T or p.S619L mutations. C25 myoblasts were transfected with IRAP-pHluorin together with alternatively WT, p.A618T or p.S619L dynamin-2-mCherry plasmids. Exocytosis induced with 10 μM ionomycin was monitored 48 h later by TIRFM. ( A ): Confocal images of cells expressing IRAP-pHluorin (green) together with WT, p.A618T or p.S619L dynamin-2 (red). Scale bar = 20 μm. ( B ): Frequency of events in C25 myoblasts overexpressing WT (black; 10 cells), p.A618T (blue; 12 cells) or p.S619L (red; 14 cells) dynamin-2 in non-stimulated conditions (empty dots) or stimulated with 10 µM ionomycin (filled dots). Each dot represents the average frequency of events for individual cells from at least three independent cultures. Horizontal lines indicate means with SEM. *** p

    Techniques Used: Transfection, Expressing

    Spontaneous and stimulus-induced fusion events in immortalized human C25 myoblasts. C25 myoblasts were transfected with IRAP-pHluorin, and exocytosis was monitored 48 h later by TIRFM. ( A ): The left panel shows a TIRFM image showing spontaneous fusion events in a non-stimulated C25 cell. Scale bar = 10 μm. The red square indicates a selected fusion event. The sequence of video frames of this event is shown in the upper right panel (every other frame was skipped). Scale bar = 0.5 μm. Time scale bar (black line) = 5.58 s. The fluorescence intensity time progress profile of this event is shown below the frame sequence. ( B ): Frequency of events in C25 myoblasts non-stimulated (N-S; 10 cells) or stimulated with 1, 5 or 10 µM ionomycin (12, 10, and 10 cells, respectively). Dots represent values obtained from individual cells from at least three independent cultures. Horizontal lines indicate means with SEM, respectively. *** p
    Figure Legend Snippet: Spontaneous and stimulus-induced fusion events in immortalized human C25 myoblasts. C25 myoblasts were transfected with IRAP-pHluorin, and exocytosis was monitored 48 h later by TIRFM. ( A ): The left panel shows a TIRFM image showing spontaneous fusion events in a non-stimulated C25 cell. Scale bar = 10 μm. The red square indicates a selected fusion event. The sequence of video frames of this event is shown in the upper right panel (every other frame was skipped). Scale bar = 0.5 μm. Time scale bar (black line) = 5.58 s. The fluorescence intensity time progress profile of this event is shown below the frame sequence. ( B ): Frequency of events in C25 myoblasts non-stimulated (N-S; 10 cells) or stimulated with 1, 5 or 10 µM ionomycin (12, 10, and 10 cells, respectively). Dots represent values obtained from individual cells from at least three independent cultures. Horizontal lines indicate means with SEM, respectively. *** p

    Techniques Used: Transfection, Sequencing, Fluorescence

    8) Product Images from "Arc ubiquitination regulates endoplasmic reticulum-mediated Ca2+ release and CaMKII signaling"

    Article Title: Arc ubiquitination regulates endoplasmic reticulum-mediated Ca2+ release and CaMKII signaling

    Journal: bioRxiv

    doi: 10.1101/2022.09.02.506383

    Baseline ER [Ca 2+ ] is not different in ArcKR primary hippocampal neurons. (A-E) Mean G-CatchER + fluorescence change from baseline (0-75 s) at the indicated regions during application of 50 μM ionomycin + 10 mM Ca 2+ to ArcWT (blue, N = 3) and ArcKR (red, N = 4) primary hippocampal neurons. Mean +/− SEM. Soma n = 3 WT, 4 KR; primary branchpoint n = 8 WT, 8 KR; primary dendrite n = 8 WT, 8 KR; secondary branchpoint n = 4 WT, 3 KR; secondary dendrites n = 3 WT, 2 KR. (F) Calculated [Ca 2+ ] ER for ArcWT and ArcKR neurons. Mean +/− SEM. Two-way ANOVA with Šídák’s multiple comparison test, no significant genotype effects. Significant simple effect of region (F (4, 41), p
    Figure Legend Snippet: Baseline ER [Ca 2+ ] is not different in ArcKR primary hippocampal neurons. (A-E) Mean G-CatchER + fluorescence change from baseline (0-75 s) at the indicated regions during application of 50 μM ionomycin + 10 mM Ca 2+ to ArcWT (blue, N = 3) and ArcKR (red, N = 4) primary hippocampal neurons. Mean +/− SEM. Soma n = 3 WT, 4 KR; primary branchpoint n = 8 WT, 8 KR; primary dendrite n = 8 WT, 8 KR; secondary branchpoint n = 4 WT, 3 KR; secondary dendrites n = 3 WT, 2 KR. (F) Calculated [Ca 2+ ] ER for ArcWT and ArcKR neurons. Mean +/− SEM. Two-way ANOVA with Šídák’s multiple comparison test, no significant genotype effects. Significant simple effect of region (F (4, 41), p

    Techniques Used: Fluorescence

    9) Product Images from "Cdk5 regulates IP3R1-mediated Ca2+ dynamics and Ca2+-mediated cell proliferation"

    Article Title: Cdk5 regulates IP3R1-mediated Ca2+ dynamics and Ca2+-mediated cell proliferation

    Journal: Cellular and Molecular Life Sciences

    doi: 10.1007/s00018-022-04515-8

    [Ca 2+ ] cyt regulates ROS production in Cdk5 −/− MEFs. Wt and Cdk5 −/− MEFs treated with 3 µM XeC, 10 µM ionomycin or 50 µM BAPTA-AM then stained with DCFDA ( A ) or mitoSOX red ( B ) were subjected to flow cytometry to measure cytoplasmic and mitochondrial ROS levels, respectively. Graphs (lower panels) show the % increase in mean fluorescence intensity. Values from wt MEFs were normalized to 1.0. Values are means ± SEM from three independent experiments ( n = 3). * p
    Figure Legend Snippet: [Ca 2+ ] cyt regulates ROS production in Cdk5 −/− MEFs. Wt and Cdk5 −/− MEFs treated with 3 µM XeC, 10 µM ionomycin or 50 µM BAPTA-AM then stained with DCFDA ( A ) or mitoSOX red ( B ) were subjected to flow cytometry to measure cytoplasmic and mitochondrial ROS levels, respectively. Graphs (lower panels) show the % increase in mean fluorescence intensity. Values from wt MEFs were normalized to 1.0. Values are means ± SEM from three independent experiments ( n = 3). * p

    Techniques Used: Staining, Flow Cytometry, Fluorescence

    10) Product Images from "TNF-α Affects Signature Cytokines of Th1 and Th17 T Cell Subsets through Differential Actions on TNFR1 and TNFR2"

    Article Title: TNF-α Affects Signature Cytokines of Th1 and Th17 T Cell Subsets through Differential Actions on TNFR1 and TNFR2

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms23169306

    Effect of TNF-α on Th1 and Th17 cells apoptosis. Purified T cell subpopulations were cultured for 4 days with 1 µg/mL of TNF-α alone or in the presence of a TNF-α neutralizing mAb (infliximab), stimulated with PMA and ionomycin plus brefeldin A for 5 h, stained with annexin-V and 7-AAD, and analyzed by flow cytometry. ( A ) Representative dot plots of the gating strategy to assess cell apoptosis. Total purified cells were analyzed. The numbers in each quadrant represent the percentage of cells. The effect of TNF-α on cell apoptosis was detected on Th1 ( B ) and Th17 ( C ) cells obtained from three healthy donors. Bars represent the mean values ± SD. Data were normalized against cells that did not receive TNF-α or TNF-α plus anti-TNF-α treatment. Statistical analyses were performed with Kruskal–Wallis followed by Dunn’s multiple comparison tests. * p
    Figure Legend Snippet: Effect of TNF-α on Th1 and Th17 cells apoptosis. Purified T cell subpopulations were cultured for 4 days with 1 µg/mL of TNF-α alone or in the presence of a TNF-α neutralizing mAb (infliximab), stimulated with PMA and ionomycin plus brefeldin A for 5 h, stained with annexin-V and 7-AAD, and analyzed by flow cytometry. ( A ) Representative dot plots of the gating strategy to assess cell apoptosis. Total purified cells were analyzed. The numbers in each quadrant represent the percentage of cells. The effect of TNF-α on cell apoptosis was detected on Th1 ( B ) and Th17 ( C ) cells obtained from three healthy donors. Bars represent the mean values ± SD. Data were normalized against cells that did not receive TNF-α or TNF-α plus anti-TNF-α treatment. Statistical analyses were performed with Kruskal–Wallis followed by Dunn’s multiple comparison tests. * p

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

    Phenotypic characterization of purified peripheral blood Th1 and Th17 cell subpopulations by flow cytometry. ( A ) A polyclonal stimulus of RosettSep™ kit-enriched CD4 + T lymphocytes with PMA and ionomycin plus anti-CD28 and anti-CD49d mAbs (pre-sorting) was followed by cell sorting of T cell subtypes based on cytokine production: IFN-γ for Th1 cells and IL-17 for Th17 cells (post-sorting). The numbers in each quadrant represent the percentage of cells. ( B ) Representative histograms showing the expression levels of the transcription factors T-bet and RORγt on purified Th1 and Th17 cells from a healthy donor. Light grey histograms, isotype control; grey histograms, Th1 cells; black histograms, Th17 cells. The numbers on top of each curve represent the mean fluorescence intensity (MFI) values. ( C ) MFI values of T-bet and RORγt levels on purified Th1 and Th17 cells obtained from four healthy donors. Bars represent the mean values ± standard deviation (SD). The Mann-Whitney test was used for statistical analysis. * p
    Figure Legend Snippet: Phenotypic characterization of purified peripheral blood Th1 and Th17 cell subpopulations by flow cytometry. ( A ) A polyclonal stimulus of RosettSep™ kit-enriched CD4 + T lymphocytes with PMA and ionomycin plus anti-CD28 and anti-CD49d mAbs (pre-sorting) was followed by cell sorting of T cell subtypes based on cytokine production: IFN-γ for Th1 cells and IL-17 for Th17 cells (post-sorting). The numbers in each quadrant represent the percentage of cells. ( B ) Representative histograms showing the expression levels of the transcription factors T-bet and RORγt on purified Th1 and Th17 cells from a healthy donor. Light grey histograms, isotype control; grey histograms, Th1 cells; black histograms, Th17 cells. The numbers on top of each curve represent the mean fluorescence intensity (MFI) values. ( C ) MFI values of T-bet and RORγt levels on purified Th1 and Th17 cells obtained from four healthy donors. Bars represent the mean values ± standard deviation (SD). The Mann-Whitney test was used for statistical analysis. * p

    Techniques Used: Purification, Flow Cytometry, FACS, Expressing, Fluorescence, Standard Deviation, MANN-WHITNEY

    Effect of TNF-α on IFN-γ or IL-17 production by Th17 lymphocytes. Sorted Th17 cells were cultured for 4 days with 1 µg/mL of TNF-α alone or in the presence of an anti-TNF-α blocking mAb (infliximab). Cells were then stimulated with PMA and ionomycin plus brefeldin A for 5 h and analyzed for cytokine production by flow cytometry. ( A ) Representative dot plots of Th17 cells upon stimulus with TNF-α. The numbers in each quadrant represent the percentage of cells. The effect of TNF-α on IFN-γ ( B ) or IL-17 ( C ) production was measured on Th17 cells from two healthy donors. Bars represent the mean values ± SD. Data were normalized against cells that did not receive TNF-α or TNF-α plus anti-TNF-α treatment. Statistical analyses were performed with Kruskal-Wallis followed by Dunn’s multiple comparison tests. * p
    Figure Legend Snippet: Effect of TNF-α on IFN-γ or IL-17 production by Th17 lymphocytes. Sorted Th17 cells were cultured for 4 days with 1 µg/mL of TNF-α alone or in the presence of an anti-TNF-α blocking mAb (infliximab). Cells were then stimulated with PMA and ionomycin plus brefeldin A for 5 h and analyzed for cytokine production by flow cytometry. ( A ) Representative dot plots of Th17 cells upon stimulus with TNF-α. The numbers in each quadrant represent the percentage of cells. The effect of TNF-α on IFN-γ ( B ) or IL-17 ( C ) production was measured on Th17 cells from two healthy donors. Bars represent the mean values ± SD. Data were normalized against cells that did not receive TNF-α or TNF-α plus anti-TNF-α treatment. Statistical analyses were performed with Kruskal-Wallis followed by Dunn’s multiple comparison tests. * p

    Techniques Used: Cell Culture, Blocking Assay, Flow Cytometry

    Effect of TNF-α on IFN-γ or IL-17 production by Th1 lymphocytes. Sorted Th1 cells were cultured for 4 days with 1 µg/mL of recombinant human TNF-α alone or in the presence of an anti-TNF-α blocking mAb (infliximab). Cells were then stimulated with PMA and ionomycin plus brefeldin A for 5 h and analyzed for cytokine production by flow cytometry. ( A ) Representative dot plots of Th1 cells upon stimulus with TNF-α. The numbers in each quadrant represent the percentage of cells. The effect of TNF-α on IFN-γ ( B ) or IL-17 ( C ) production was measured on Th1 cells from three healthy donors. Bars represent the mean values ± SD. Data were normalized against cells that did not receive TNF-α or TNF-α plus anti-TNF-α treatment. Kruskal–Wallis test followed by Dunn’s multiple comparison test was carried out to analyze differences between groups. * p
    Figure Legend Snippet: Effect of TNF-α on IFN-γ or IL-17 production by Th1 lymphocytes. Sorted Th1 cells were cultured for 4 days with 1 µg/mL of recombinant human TNF-α alone or in the presence of an anti-TNF-α blocking mAb (infliximab). Cells were then stimulated with PMA and ionomycin plus brefeldin A for 5 h and analyzed for cytokine production by flow cytometry. ( A ) Representative dot plots of Th1 cells upon stimulus with TNF-α. The numbers in each quadrant represent the percentage of cells. The effect of TNF-α on IFN-γ ( B ) or IL-17 ( C ) production was measured on Th1 cells from three healthy donors. Bars represent the mean values ± SD. Data were normalized against cells that did not receive TNF-α or TNF-α plus anti-TNF-α treatment. Kruskal–Wallis test followed by Dunn’s multiple comparison test was carried out to analyze differences between groups. * p

    Techniques Used: Cell Culture, Recombinant, Blocking Assay, Flow Cytometry

    11) Product Images from "PLXND1-mediated calcium dyshomeostasis impairs endocardial endothelial autophagy in atrial fibrillation"

    Article Title: PLXND1-mediated calcium dyshomeostasis impairs endocardial endothelial autophagy in atrial fibrillation

    Journal: Frontiers in Physiology

    doi: 10.3389/fphys.2022.960480

    PLXND1 inhibits autophagy via intracellular calcium signaling. Mouse EECs were treated with NC, LV- Plxnd1 , and sh Plxnd1 . (A) Representative WB of PLXND1 in EECs subjected to the indicated treatments. (B) Quantitative analysis of the efficacy of overexpression and knockdown of Plxnd1 . (C) Representative western blots of autophagic markers in EECs subjected to the indicated treatments. (D–E) Quantitative analysis indicates that PLXND1 significantly affects the level of autophagy. (F) Immunofluorescence staining was used to detect MAP1LC3B in EECs. Scale bar: 20 μm. (G) Intracellular calcium was marked by the Fluo-3-AM probe and measured continuously for 8 min (H) EECs were incubated with the calcium probe Fluo-3-AM. EGTA + BAPTA-AM was used to obtain the minimum fluorescence intensity (F min ), whereas ionomycin + calcium was used to obtain the maximum fluorescence intensity (F max ). Scale bar: 20 μm. Quantitative analysis showed the fluorescence intensity in the different groups. (I-J) EECs were infected with a tandem GFP-mRFP-LC3 adenovirus for 24 h. Representative merged images (I) and quantitative analysis (J) of yellow and free red puncta in merged images of different treatment groups. Scale bar: 10 μm. EECs, endocardial endothelial cells; NC, negative control; PLXND1, plexinD1; WB, western blotting; EGTA, ethylene glycol tetraacetic acid.
    Figure Legend Snippet: PLXND1 inhibits autophagy via intracellular calcium signaling. Mouse EECs were treated with NC, LV- Plxnd1 , and sh Plxnd1 . (A) Representative WB of PLXND1 in EECs subjected to the indicated treatments. (B) Quantitative analysis of the efficacy of overexpression and knockdown of Plxnd1 . (C) Representative western blots of autophagic markers in EECs subjected to the indicated treatments. (D–E) Quantitative analysis indicates that PLXND1 significantly affects the level of autophagy. (F) Immunofluorescence staining was used to detect MAP1LC3B in EECs. Scale bar: 20 μm. (G) Intracellular calcium was marked by the Fluo-3-AM probe and measured continuously for 8 min (H) EECs were incubated with the calcium probe Fluo-3-AM. EGTA + BAPTA-AM was used to obtain the minimum fluorescence intensity (F min ), whereas ionomycin + calcium was used to obtain the maximum fluorescence intensity (F max ). Scale bar: 20 μm. Quantitative analysis showed the fluorescence intensity in the different groups. (I-J) EECs were infected with a tandem GFP-mRFP-LC3 adenovirus for 24 h. Representative merged images (I) and quantitative analysis (J) of yellow and free red puncta in merged images of different treatment groups. Scale bar: 10 μm. EECs, endocardial endothelial cells; NC, negative control; PLXND1, plexinD1; WB, western blotting; EGTA, ethylene glycol tetraacetic acid.

    Techniques Used: Western Blot, Over Expression, Immunofluorescence, Staining, Incubation, Fluorescence, Infection, Negative Control

    12) Product Images from "Lack of Oestrogen Receptor Expression in Breast Cancer Cells Does Not Correlate with Kisspeptin Signalling and Migration"

    Article Title: Lack of Oestrogen Receptor Expression in Breast Cancer Cells Does Not Correlate with Kisspeptin Signalling and Migration

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms23158744

    KP-10 induces similar levels of calcium mobilization in BT-20 and MDA-MB-231 cells. BT-20 and MDA-MB-231 cells were loaded with the calcium indicator dye, Fluo-3 AM, imaged using a Zeiss LSM800 at 20× magnification every 1.5 s before 100 nM KP-10 was added. After at least 300 s, 1 µM Ionomycin + 1 M CaCl 2 was added (positive control) to determine the maximal Fluo-3 excitation. ( a ) Fluorescence images of BT-20 and MDA-MB-231 cells before (-) and after KP-10 stimulation. ( b ) A representative experiment showing 20 BT-20 cells tracked for fluorescence intensity before and after KP-10 stimulation (arrow) and Ionomycin stimulation (star). ( c ) A representative experiment showing 20 MDA-MB-231 cells tracked for fluorescence intensity before and after KP-10 stimulation (arrow) and Ionomycin stimulation (star). ( d ) Quantification (mean ± SEM) of the maximum amplitude reached after KP-10 stimulation in BT-20 and MDA-MB-231 cells ( e ) Quantification (mean ± SEM) of the time taken to reach maximum mobilization after KP-10 stimulation in BT-20 and MDA-MB-231 cells. There was no significant difference ( p > 0.05) in maximum amplitude or time to maximum amplitude between the two cell lines.
    Figure Legend Snippet: KP-10 induces similar levels of calcium mobilization in BT-20 and MDA-MB-231 cells. BT-20 and MDA-MB-231 cells were loaded with the calcium indicator dye, Fluo-3 AM, imaged using a Zeiss LSM800 at 20× magnification every 1.5 s before 100 nM KP-10 was added. After at least 300 s, 1 µM Ionomycin + 1 M CaCl 2 was added (positive control) to determine the maximal Fluo-3 excitation. ( a ) Fluorescence images of BT-20 and MDA-MB-231 cells before (-) and after KP-10 stimulation. ( b ) A representative experiment showing 20 BT-20 cells tracked for fluorescence intensity before and after KP-10 stimulation (arrow) and Ionomycin stimulation (star). ( c ) A representative experiment showing 20 MDA-MB-231 cells tracked for fluorescence intensity before and after KP-10 stimulation (arrow) and Ionomycin stimulation (star). ( d ) Quantification (mean ± SEM) of the maximum amplitude reached after KP-10 stimulation in BT-20 and MDA-MB-231 cells ( e ) Quantification (mean ± SEM) of the time taken to reach maximum mobilization after KP-10 stimulation in BT-20 and MDA-MB-231 cells. There was no significant difference ( p > 0.05) in maximum amplitude or time to maximum amplitude between the two cell lines.

    Techniques Used: Multiple Displacement Amplification, Positive Control, Fluorescence

    13) Product Images from "A public antibody class recognizes an S2 epitope exposed on open conformations of SARS-CoV-2 spike"

    Article Title: A public antibody class recognizes an S2 epitope exposed on open conformations of SARS-CoV-2 spike

    Journal: Nature Communications

    doi: 10.1038/s41467-022-32232-0

    Genotypic and phenotypic characterization of SARS-CoV-2 S-reactive monoclonal antibodies from unexposed individuals. A Representative gating strategy of SARS-CoV-2 S-reactive B cells. Antigen-reactive B cells, were associated to naïve, unswitched IgD + memory, CD27- memory, and classical memory phenotype) (lower panel). B Dot plot overlaying a violin plot showing sequence identity (%) to IMGT-annotated germline heavy chain sequences for all isolated BCR heavy chains. Each color represents a B cell phenotype from four healthy donors. HD, healthy donor. C Bar plot showing the number of sequences recovered for each immunoglobulin heavy chain V (IGHV, left panel) gene and immunoglobulin kappa/lambda light chain (IGKV/IGLV, right panel) gene. Colored bars represent different HDs. D Matrix showing the number of pairs with a certain IGHV (x-axis) and IGKV ( y -axis). The numbers inside the boxes represent the number of pairs recovered for each pairing. E Matrix showing flow cytometric binding assay to SARS-CoV-2 S-transfected or untransfected HEK293T cells for each selected MAb and control MAb COVA1-18. Numbers and colors in the boxes represent the percentage of cells showing binding to a particular MAb. The phenotype as determined in ( A ) is shown on top of the matrix. F Matrix depicting area under the curve (AUC) as determined by a polyreactive enzyme-linked immunosorbent assay (ELISA) for each of the antigens shown on the left. The letters on top represent B cell phenotypes as determined in ( A ). Polyreactive MAbs are indicated in bold text. nd not determined. G Antigen specificity of Ramos B cells designed to express a 1C12, 3C9, PGT121 or COVA2-15 BCR to SARS-CoV-2 S (left) or HIV-1 Env (right). The numbers inside the boxes represent the frequency (%) of cells in a gate. H Ramos B cell activation of 1C12 B cells (top panel) and 3C9 B cells (bottom panel) as measured by calcium (Ca 2+ ) flux assay. A baseline without antigen was established between 0 and 30 s, after which the measurement was interrupted to add the antigen to the B cells (30–50 s). Ionomycin was used at 1 μg/mL as positive control.
    Figure Legend Snippet: Genotypic and phenotypic characterization of SARS-CoV-2 S-reactive monoclonal antibodies from unexposed individuals. A Representative gating strategy of SARS-CoV-2 S-reactive B cells. Antigen-reactive B cells, were associated to naïve, unswitched IgD + memory, CD27- memory, and classical memory phenotype) (lower panel). B Dot plot overlaying a violin plot showing sequence identity (%) to IMGT-annotated germline heavy chain sequences for all isolated BCR heavy chains. Each color represents a B cell phenotype from four healthy donors. HD, healthy donor. C Bar plot showing the number of sequences recovered for each immunoglobulin heavy chain V (IGHV, left panel) gene and immunoglobulin kappa/lambda light chain (IGKV/IGLV, right panel) gene. Colored bars represent different HDs. D Matrix showing the number of pairs with a certain IGHV (x-axis) and IGKV ( y -axis). The numbers inside the boxes represent the number of pairs recovered for each pairing. E Matrix showing flow cytometric binding assay to SARS-CoV-2 S-transfected or untransfected HEK293T cells for each selected MAb and control MAb COVA1-18. Numbers and colors in the boxes represent the percentage of cells showing binding to a particular MAb. The phenotype as determined in ( A ) is shown on top of the matrix. F Matrix depicting area under the curve (AUC) as determined by a polyreactive enzyme-linked immunosorbent assay (ELISA) for each of the antigens shown on the left. The letters on top represent B cell phenotypes as determined in ( A ). Polyreactive MAbs are indicated in bold text. nd not determined. G Antigen specificity of Ramos B cells designed to express a 1C12, 3C9, PGT121 or COVA2-15 BCR to SARS-CoV-2 S (left) or HIV-1 Env (right). The numbers inside the boxes represent the frequency (%) of cells in a gate. H Ramos B cell activation of 1C12 B cells (top panel) and 3C9 B cells (bottom panel) as measured by calcium (Ca 2+ ) flux assay. A baseline without antigen was established between 0 and 30 s, after which the measurement was interrupted to add the antigen to the B cells (30–50 s). Ionomycin was used at 1 μg/mL as positive control.

    Techniques Used: Sequencing, Isolation, Binding Assay, Transfection, Enzyme-linked Immunosorbent Assay, Activation Assay, Flux Assay, Positive Control

    14) Product Images from "Adoptive Cellular Therapy with Autologous Tumor-Infiltrating Lymphocytes and T-cell Receptor-Engineered T Cells Targeting Common p53 Neoantigens in Human Solid Tumors"

    Article Title: Adoptive Cellular Therapy with Autologous Tumor-Infiltrating Lymphocytes and T-cell Receptor-Engineered T Cells Targeting Common p53 Neoantigens in Human Solid Tumors

    Journal: Cancer immunology research

    doi: 10.1158/2326-6066.CIR-22-0040

    Unbiased neoantigen screening of TILs from patient 4316 identifies TILs/TCR reactive with mutant p53 C135Y . A, Diagram depicting the unbiased neoantigen screening for immunogenicity of shared TP53 mutations. B, ELISpot assay measuring IFNg secretion in TILs. The 24 TIL fragment subcultures from colorectal cancer patient 4316 were screened against the somatic mutations identified in the patient’s tumor, including p53 C135Y . TIL fragment 22 showing increased IFNγ secretion against TMGs and PPs that included p53 C135Y is highlighted in red ( n = 1). C, Flow cytometric analysis of cell surface 4–1BB/OX40 expression upon parsing individual reactivities of TIL fragment 22. An irrelevant peptide (KIAA1328 K386R ) and DMSO (vehicle) as negative controls and PMA/ionomycin as a positive control are included ( n = 1). D, Functional determination of MEs for TCR-B by ELISpot measurement of IFNγ secretion. Eighteen candidate MEs predicted to bind patient’s class I HLAs were tested. The amino acid sequences of the tested peptides are listed ( n = 1). E, Quantification of IFNγ spots from panel ( D ). F, Functional testing of avidity of TCR-B. Avidity was determined by coculturing TCR-expressing healthy donor PBLs with autologous imDCs pulsed with serially diluted ME6. 4–1BB expression was measured by flow cytometry ( n = 1). G, Autologous tumor cell recognition by 4316 TCR-B. The 4316 autologous PDX was established into a cell line and was cocultured with TCR-B–expressing PBLs. 4–1BB upregulation in p53 C135Y -TCR + CD8 + cells is shown. The 4247 PDX line with matching HLA but lacking a p53 C135Y mutation was included as a negative control. ( n = 1) Experiments in ( B ) to ( G ) were independently repeated once. seq, sequencing. PP, peptide pool.
    Figure Legend Snippet: Unbiased neoantigen screening of TILs from patient 4316 identifies TILs/TCR reactive with mutant p53 C135Y . A, Diagram depicting the unbiased neoantigen screening for immunogenicity of shared TP53 mutations. B, ELISpot assay measuring IFNg secretion in TILs. The 24 TIL fragment subcultures from colorectal cancer patient 4316 were screened against the somatic mutations identified in the patient’s tumor, including p53 C135Y . TIL fragment 22 showing increased IFNγ secretion against TMGs and PPs that included p53 C135Y is highlighted in red ( n = 1). C, Flow cytometric analysis of cell surface 4–1BB/OX40 expression upon parsing individual reactivities of TIL fragment 22. An irrelevant peptide (KIAA1328 K386R ) and DMSO (vehicle) as negative controls and PMA/ionomycin as a positive control are included ( n = 1). D, Functional determination of MEs for TCR-B by ELISpot measurement of IFNγ secretion. Eighteen candidate MEs predicted to bind patient’s class I HLAs were tested. The amino acid sequences of the tested peptides are listed ( n = 1). E, Quantification of IFNγ spots from panel ( D ). F, Functional testing of avidity of TCR-B. Avidity was determined by coculturing TCR-expressing healthy donor PBLs with autologous imDCs pulsed with serially diluted ME6. 4–1BB expression was measured by flow cytometry ( n = 1). G, Autologous tumor cell recognition by 4316 TCR-B. The 4316 autologous PDX was established into a cell line and was cocultured with TCR-B–expressing PBLs. 4–1BB upregulation in p53 C135Y -TCR + CD8 + cells is shown. The 4247 PDX line with matching HLA but lacking a p53 C135Y mutation was included as a negative control. ( n = 1) Experiments in ( B ) to ( G ) were independently repeated once. seq, sequencing. PP, peptide pool.

    Techniques Used: Mutagenesis, Enzyme-linked Immunospot, Expressing, Positive Control, Functional Assay, Flow Cytometry, Negative Control, Sequencing

    15) Product Images from "Store‐operated calcium entry controls innate and adaptive immune cell function in inflammatory bowel disease"

    Article Title: Store‐operated calcium entry controls innate and adaptive immune cell function in inflammatory bowel disease

    Journal: EMBO Molecular Medicine

    doi: 10.15252/emmm.202215687

    Four clusters of myeloid cells in the colon lamina propria (CLP) of IBD patients and effects of SOCE inhibition (Left) FlowSOM plot of merged FCS files from unstimulated IBD samples and samples treated with PMA/ionomycin ± 1 μM BTP2 (non‐inflamed: n = 4, CD: n = 6, UC: n = 6). Colors indicate myeloid cell clusters within CD45 + CD3 − LPMCs. (Right) viSNE plots of one exemplary CD patient. Colors indicate expression levels of cell surface markers (blue: low; red: high) in cells left unstimulated, stimulated with PMA/ionomycin or stimulated in the presence of 1 μM BTP2.
    Figure Legend Snippet: Four clusters of myeloid cells in the colon lamina propria (CLP) of IBD patients and effects of SOCE inhibition (Left) FlowSOM plot of merged FCS files from unstimulated IBD samples and samples treated with PMA/ionomycin ± 1 μM BTP2 (non‐inflamed: n = 4, CD: n = 6, UC: n = 6). Colors indicate myeloid cell clusters within CD45 + CD3 − LPMCs. (Right) viSNE plots of one exemplary CD patient. Colors indicate expression levels of cell surface markers (blue: low; red: high) in cells left unstimulated, stimulated with PMA/ionomycin or stimulated in the presence of 1 μM BTP2.

    Techniques Used: Inhibition, Expressing

    Deletion of SOCE in CD4 + T cells suppresses the induction of colitis in mice Naïve CD4 + T cells were isolated from the spleen and LNs of WT, Stim2 fl/fl Cd4Cre (S2), Orai1 fl/fl Cd4Cre (O1), and Stim1 fl/fl Cd4Cre (S1) mice and differentiated into Th1, Th17, and iTreg cells. (A) Fura‐2 loaded cells were stimulated with 0.3 μM ionomycin to induce SOCE. Representative Ca 2+ traces (left) and peak Ca 2+ levels (right). Data are from the mean ± SEM of four mice per group. (B) Induction of colitis in Rag1 −/− mice by adoptive transfer of naive CD4 + T cells from wildtype, Stim2 fl/fl Cd4Cre , Orai1 fl/fl Cd4Cre and Stim1 fl/fl Cd4Cre mice. Weight curves of recipient Rag1 −/− mice following injection of naive CD4 + T cells. Data are from two independent experiments with eight recipient mice per group. (C) H E stains of colon tissues and histological inflammation scores of Rag1 −/− mice 8 weeks after T cell transfer. Data are from one representative experiment with five mice per group. (D, E) Representative flow cytometry plots and frequencies of donor CD4 + T cells producing IFNγ, IL‐17A, TNF or expressing Foxp3 that were isolated from the mesenteric LNs of recipient Rag1 −/− mice. Data are from two independent experiments with 3–8 mice per group. Statistical analyses by unpaired student's t ‐test: *** P
    Figure Legend Snippet: Deletion of SOCE in CD4 + T cells suppresses the induction of colitis in mice Naïve CD4 + T cells were isolated from the spleen and LNs of WT, Stim2 fl/fl Cd4Cre (S2), Orai1 fl/fl Cd4Cre (O1), and Stim1 fl/fl Cd4Cre (S1) mice and differentiated into Th1, Th17, and iTreg cells. (A) Fura‐2 loaded cells were stimulated with 0.3 μM ionomycin to induce SOCE. Representative Ca 2+ traces (left) and peak Ca 2+ levels (right). Data are from the mean ± SEM of four mice per group. (B) Induction of colitis in Rag1 −/− mice by adoptive transfer of naive CD4 + T cells from wildtype, Stim2 fl/fl Cd4Cre , Orai1 fl/fl Cd4Cre and Stim1 fl/fl Cd4Cre mice. Weight curves of recipient Rag1 −/− mice following injection of naive CD4 + T cells. Data are from two independent experiments with eight recipient mice per group. (C) H E stains of colon tissues and histological inflammation scores of Rag1 −/− mice 8 weeks after T cell transfer. Data are from one representative experiment with five mice per group. (D, E) Representative flow cytometry plots and frequencies of donor CD4 + T cells producing IFNγ, IL‐17A, TNF or expressing Foxp3 that were isolated from the mesenteric LNs of recipient Rag1 −/− mice. Data are from two independent experiments with 3–8 mice per group. Statistical analyses by unpaired student's t ‐test: *** P

    Techniques Used: Mouse Assay, Isolation, Adoptive Transfer Assay, Injection, Flow Cytometry, Expressing

    Inhibition of SOCE suppresses the activation and function of human colonic T cells FlowSOM plot of merged FCS files from samples treated with PMA/ionomycin ± 1 μM BTP2 (non‐inflamed: n = 4, CD: n = 6, UC: n = 6). Colors of FlowSOM plot indicate 20 distinct clusters of CD45 + CD3 + LPMCs. Heatmaps show the expression levels of 24 markers used for cluster determination. The table shows the mean ± SEM of frequencies (%) for each cell subset defined in non‐inflamed controls, UC and CD patients after 4 h stimulation with PMA/ionomycin in vitro . Statistical significance was calculated using the edgeR statistical framework with negative binomial GLM and a false discovery rate adjusted to 10% using the Benjiamini–Hochberg procedure. Box plots showing frequencies (%) of each cell subset defined by the cluster analysis in non‐inflamed, UC and CD samples following stimulation with PMA/ionomycin ± BTP2 for 4 h in vitro . Statistical significances were calculated using a one‐tailed paired Wilcoxon matched‐pairs signed‐rank test, * P
    Figure Legend Snippet: Inhibition of SOCE suppresses the activation and function of human colonic T cells FlowSOM plot of merged FCS files from samples treated with PMA/ionomycin ± 1 μM BTP2 (non‐inflamed: n = 4, CD: n = 6, UC: n = 6). Colors of FlowSOM plot indicate 20 distinct clusters of CD45 + CD3 + LPMCs. Heatmaps show the expression levels of 24 markers used for cluster determination. The table shows the mean ± SEM of frequencies (%) for each cell subset defined in non‐inflamed controls, UC and CD patients after 4 h stimulation with PMA/ionomycin in vitro . Statistical significance was calculated using the edgeR statistical framework with negative binomial GLM and a false discovery rate adjusted to 10% using the Benjiamini–Hochberg procedure. Box plots showing frequencies (%) of each cell subset defined by the cluster analysis in non‐inflamed, UC and CD samples following stimulation with PMA/ionomycin ± BTP2 for 4 h in vitro . Statistical significances were calculated using a one‐tailed paired Wilcoxon matched‐pairs signed‐rank test, * P

    Techniques Used: Inhibition, Activation Assay, Expressing, In Vitro, One-tailed Test

    Effects of SOCE inhibition on CD3 − lamina propria cells FlowSOM plot of merged FCS files from unstimulated samples or samples treated with PMA/ionomycin ± 1 μM BTP2 (non‐inflamed: n = 4, CD: n = 6, UC: n = 6). Colors of FlowSOM plot indicate 20 distinct clusters of CD45 + CD3 − LPMCs. Heatmap clusters showing the expression levels of 34 markers used for cluster determination. The table shows the mean ± SEM of frequencies (%) for each cell subset defined in non‐Inflamed, UC, and CD samples after stimulation with PMA/ionomycin for 4 h. Statistical significance was calculated using the edgeR statistical framework with negative binomial GLM and a false discovery rate adjusted to 10% using the Benjiamini–Hochberg procedure. Box plots show frequencies (%) of each cell subset defined by the cluster analysis in samples of non‐inflamed controls, UC or CD patients after stimulation with PMA/ionomycin ± BTP2 for 4 h ex vivo . Statistical significances were calculated by one‐tailed paired Wilcoxon matched‐pairs signed‐rank test, * P
    Figure Legend Snippet: Effects of SOCE inhibition on CD3 − lamina propria cells FlowSOM plot of merged FCS files from unstimulated samples or samples treated with PMA/ionomycin ± 1 μM BTP2 (non‐inflamed: n = 4, CD: n = 6, UC: n = 6). Colors of FlowSOM plot indicate 20 distinct clusters of CD45 + CD3 − LPMCs. Heatmap clusters showing the expression levels of 34 markers used for cluster determination. The table shows the mean ± SEM of frequencies (%) for each cell subset defined in non‐Inflamed, UC, and CD samples after stimulation with PMA/ionomycin for 4 h. Statistical significance was calculated using the edgeR statistical framework with negative binomial GLM and a false discovery rate adjusted to 10% using the Benjiamini–Hochberg procedure. Box plots show frequencies (%) of each cell subset defined by the cluster analysis in samples of non‐inflamed controls, UC or CD patients after stimulation with PMA/ionomycin ± BTP2 for 4 h ex vivo . Statistical significances were calculated by one‐tailed paired Wilcoxon matched‐pairs signed‐rank test, * P

    Techniques Used: Inhibition, Expressing, Ex Vivo, One-tailed Test

    Effects of PMA/ionomycin stimulation on protein expression of human lamina propria (LP) immune cells Heatmaps representing the median fold change (FC) of cytokine and surface marker expression in CD45 + CD3 + T cells (top row) and CD45 + CD3 − immune cells (bottom row) isolated from colon lamina propria of five patients with Crohn's disease. Cells were treated with 20 ng/ml PMA and 1 μg/m (P/I) or DMSO (unstimulated) for 4 h in vitro , and protein expression was measured by mass cytometry (CyTOF). FC values are relative to unstimulated samples. Source data are available online for this figure.
    Figure Legend Snippet: Effects of PMA/ionomycin stimulation on protein expression of human lamina propria (LP) immune cells Heatmaps representing the median fold change (FC) of cytokine and surface marker expression in CD45 + CD3 + T cells (top row) and CD45 + CD3 − immune cells (bottom row) isolated from colon lamina propria of five patients with Crohn's disease. Cells were treated with 20 ng/ml PMA and 1 μg/m (P/I) or DMSO (unstimulated) for 4 h in vitro , and protein expression was measured by mass cytometry (CyTOF). FC values are relative to unstimulated samples. Source data are available online for this figure.

    Techniques Used: Expressing, Marker, Isolation, In Vitro, Mass Cytometry

    Global mass cytometric analysis of the immune cell composition in the colonic lamina propria of therapy refractory IBD patients Experimental setup for mass cytometric assays. Non‐inflamed: n = 4, CD: n = 6, UC: n = 6. FlowSOM plot of merged FCS files from LPMCs of non‐inflamed controls, UC or CD patients after stimulation with PMA/ionomycin ± 1 μM BTP2 for 4 h ex vivo . Colors of the t‐SNE plot represent 20 clusters of distinct CD45 + LPMC lineages. The heatmap shows the expression levels of 21 markers used for defining cell clusters. FlowSOM map of merged FCS files from CD45 + cells of non‐inflamed controls, UC and CD patients that were left unstimulated or treated with PMA/ionomycin ± 1 μM BTP2. Colors indicate altered cell clusters in CD patients vs. controls, UC patients vs. controls and CD vs. UC patients. viSNE plots of one exemplary non‐inflamed control, UC and CD patient colored by marker expression levels (blue: low, red: high). Quantified frequencies (%) of each cell subset defined by the cluster analysis. Statistical significance was calculated using the edgeR statistical framework with negative binomial GLM and a false discovery rate adjusted to 10% using the Benjiamini–Hochberg procedure (* P adj
    Figure Legend Snippet: Global mass cytometric analysis of the immune cell composition in the colonic lamina propria of therapy refractory IBD patients Experimental setup for mass cytometric assays. Non‐inflamed: n = 4, CD: n = 6, UC: n = 6. FlowSOM plot of merged FCS files from LPMCs of non‐inflamed controls, UC or CD patients after stimulation with PMA/ionomycin ± 1 μM BTP2 for 4 h ex vivo . Colors of the t‐SNE plot represent 20 clusters of distinct CD45 + LPMC lineages. The heatmap shows the expression levels of 21 markers used for defining cell clusters. FlowSOM map of merged FCS files from CD45 + cells of non‐inflamed controls, UC and CD patients that were left unstimulated or treated with PMA/ionomycin ± 1 μM BTP2. Colors indicate altered cell clusters in CD patients vs. controls, UC patients vs. controls and CD vs. UC patients. viSNE plots of one exemplary non‐inflamed control, UC and CD patient colored by marker expression levels (blue: low, red: high). Quantified frequencies (%) of each cell subset defined by the cluster analysis. Statistical significance was calculated using the edgeR statistical framework with negative binomial GLM and a false discovery rate adjusted to 10% using the Benjiamini–Hochberg procedure (* P adj

    Techniques Used: Ex Vivo, Expressing, Marker

    16) Product Images from "Store‐operated calcium entry controls innate and adaptive immune cell function in inflammatory bowel disease"

    Article Title: Store‐operated calcium entry controls innate and adaptive immune cell function in inflammatory bowel disease

    Journal: EMBO Molecular Medicine

    doi: 10.15252/emmm.202215687

    Four clusters of myeloid cells in the colon lamina propria (CLP) of IBD patients and effects of SOCE inhibition (Left) FlowSOM plot of merged FCS files from unstimulated IBD samples and samples treated with PMA/ionomycin ± 1 μM BTP2 (non‐inflamed: n = 4, CD: n = 6, UC: n = 6). Colors indicate myeloid cell clusters within CD45 + CD3 − LPMCs. (Right) viSNE plots of one exemplary CD patient. Colors indicate expression levels of cell surface markers (blue: low; red: high) in cells left unstimulated, stimulated with PMA/ionomycin or stimulated in the presence of 1 μM BTP2.
    Figure Legend Snippet: Four clusters of myeloid cells in the colon lamina propria (CLP) of IBD patients and effects of SOCE inhibition (Left) FlowSOM plot of merged FCS files from unstimulated IBD samples and samples treated with PMA/ionomycin ± 1 μM BTP2 (non‐inflamed: n = 4, CD: n = 6, UC: n = 6). Colors indicate myeloid cell clusters within CD45 + CD3 − LPMCs. (Right) viSNE plots of one exemplary CD patient. Colors indicate expression levels of cell surface markers (blue: low; red: high) in cells left unstimulated, stimulated with PMA/ionomycin or stimulated in the presence of 1 μM BTP2.

    Techniques Used: Inhibition, Expressing

    Deletion of SOCE in CD4 + T cells suppresses the induction of colitis in mice Naïve CD4 + T cells were isolated from the spleen and LNs of WT, Stim2 fl/fl Cd4Cre (S2), Orai1 fl/fl Cd4Cre (O1), and Stim1 fl/fl Cd4Cre (S1) mice and differentiated into Th1, Th17, and iTreg cells. (A) Fura‐2 loaded cells were stimulated with 0.3 μM ionomycin to induce SOCE. Representative Ca 2+ traces (left) and peak Ca 2+ levels (right). Data are from the mean ± SEM of four mice per group. (B) Induction of colitis in Rag1 −/− mice by adoptive transfer of naive CD4 + T cells from wildtype, Stim2 fl/fl Cd4Cre , Orai1 fl/fl Cd4Cre and Stim1 fl/fl Cd4Cre mice. Weight curves of recipient Rag1 −/− mice following injection of naive CD4 + T cells. Data are from two independent experiments with eight recipient mice per group. (C) H E stains of colon tissues and histological inflammation scores of Rag1 −/− mice 8 weeks after T cell transfer. Data are from one representative experiment with five mice per group. (D, E) Representative flow cytometry plots and frequencies of donor CD4 + T cells producing IFNγ, IL‐17A, TNF or expressing Foxp3 that were isolated from the mesenteric LNs of recipient Rag1 −/− mice. Data are from two independent experiments with 3–8 mice per group. Statistical analyses by unpaired student's t ‐test: *** P
    Figure Legend Snippet: Deletion of SOCE in CD4 + T cells suppresses the induction of colitis in mice Naïve CD4 + T cells were isolated from the spleen and LNs of WT, Stim2 fl/fl Cd4Cre (S2), Orai1 fl/fl Cd4Cre (O1), and Stim1 fl/fl Cd4Cre (S1) mice and differentiated into Th1, Th17, and iTreg cells. (A) Fura‐2 loaded cells were stimulated with 0.3 μM ionomycin to induce SOCE. Representative Ca 2+ traces (left) and peak Ca 2+ levels (right). Data are from the mean ± SEM of four mice per group. (B) Induction of colitis in Rag1 −/− mice by adoptive transfer of naive CD4 + T cells from wildtype, Stim2 fl/fl Cd4Cre , Orai1 fl/fl Cd4Cre and Stim1 fl/fl Cd4Cre mice. Weight curves of recipient Rag1 −/− mice following injection of naive CD4 + T cells. Data are from two independent experiments with eight recipient mice per group. (C) H E stains of colon tissues and histological inflammation scores of Rag1 −/− mice 8 weeks after T cell transfer. Data are from one representative experiment with five mice per group. (D, E) Representative flow cytometry plots and frequencies of donor CD4 + T cells producing IFNγ, IL‐17A, TNF or expressing Foxp3 that were isolated from the mesenteric LNs of recipient Rag1 −/− mice. Data are from two independent experiments with 3–8 mice per group. Statistical analyses by unpaired student's t ‐test: *** P

    Techniques Used: Mouse Assay, Isolation, Adoptive Transfer Assay, Injection, Flow Cytometry, Expressing

    Inhibition of SOCE suppresses the activation and function of human colonic T cells FlowSOM plot of merged FCS files from samples treated with PMA/ionomycin ± 1 μM BTP2 (non‐inflamed: n = 4, CD: n = 6, UC: n = 6). Colors of FlowSOM plot indicate 20 distinct clusters of CD45 + CD3 + LPMCs. Heatmaps show the expression levels of 24 markers used for cluster determination. The table shows the mean ± SEM of frequencies (%) for each cell subset defined in non‐inflamed controls, UC and CD patients after 4 h stimulation with PMA/ionomycin in vitro . Statistical significance was calculated using the edgeR statistical framework with negative binomial GLM and a false discovery rate adjusted to 10% using the Benjiamini–Hochberg procedure. Box plots showing frequencies (%) of each cell subset defined by the cluster analysis in non‐inflamed, UC and CD samples following stimulation with PMA/ionomycin ± BTP2 for 4 h in vitro . Statistical significances were calculated using a one‐tailed paired Wilcoxon matched‐pairs signed‐rank test, * P
    Figure Legend Snippet: Inhibition of SOCE suppresses the activation and function of human colonic T cells FlowSOM plot of merged FCS files from samples treated with PMA/ionomycin ± 1 μM BTP2 (non‐inflamed: n = 4, CD: n = 6, UC: n = 6). Colors of FlowSOM plot indicate 20 distinct clusters of CD45 + CD3 + LPMCs. Heatmaps show the expression levels of 24 markers used for cluster determination. The table shows the mean ± SEM of frequencies (%) for each cell subset defined in non‐inflamed controls, UC and CD patients after 4 h stimulation with PMA/ionomycin in vitro . Statistical significance was calculated using the edgeR statistical framework with negative binomial GLM and a false discovery rate adjusted to 10% using the Benjiamini–Hochberg procedure. Box plots showing frequencies (%) of each cell subset defined by the cluster analysis in non‐inflamed, UC and CD samples following stimulation with PMA/ionomycin ± BTP2 for 4 h in vitro . Statistical significances were calculated using a one‐tailed paired Wilcoxon matched‐pairs signed‐rank test, * P

    Techniques Used: Inhibition, Activation Assay, Expressing, In Vitro, One-tailed Test

    Effects of SOCE inhibition on CD3 − lamina propria cells FlowSOM plot of merged FCS files from unstimulated samples or samples treated with PMA/ionomycin ± 1 μM BTP2 (non‐inflamed: n = 4, CD: n = 6, UC: n = 6). Colors of FlowSOM plot indicate 20 distinct clusters of CD45 + CD3 − LPMCs. Heatmap clusters showing the expression levels of 34 markers used for cluster determination. The table shows the mean ± SEM of frequencies (%) for each cell subset defined in non‐Inflamed, UC, and CD samples after stimulation with PMA/ionomycin for 4 h. Statistical significance was calculated using the edgeR statistical framework with negative binomial GLM and a false discovery rate adjusted to 10% using the Benjiamini–Hochberg procedure. Box plots show frequencies (%) of each cell subset defined by the cluster analysis in samples of non‐inflamed controls, UC or CD patients after stimulation with PMA/ionomycin ± BTP2 for 4 h ex vivo . Statistical significances were calculated by one‐tailed paired Wilcoxon matched‐pairs signed‐rank test, * P
    Figure Legend Snippet: Effects of SOCE inhibition on CD3 − lamina propria cells FlowSOM plot of merged FCS files from unstimulated samples or samples treated with PMA/ionomycin ± 1 μM BTP2 (non‐inflamed: n = 4, CD: n = 6, UC: n = 6). Colors of FlowSOM plot indicate 20 distinct clusters of CD45 + CD3 − LPMCs. Heatmap clusters showing the expression levels of 34 markers used for cluster determination. The table shows the mean ± SEM of frequencies (%) for each cell subset defined in non‐Inflamed, UC, and CD samples after stimulation with PMA/ionomycin for 4 h. Statistical significance was calculated using the edgeR statistical framework with negative binomial GLM and a false discovery rate adjusted to 10% using the Benjiamini–Hochberg procedure. Box plots show frequencies (%) of each cell subset defined by the cluster analysis in samples of non‐inflamed controls, UC or CD patients after stimulation with PMA/ionomycin ± BTP2 for 4 h ex vivo . Statistical significances were calculated by one‐tailed paired Wilcoxon matched‐pairs signed‐rank test, * P

    Techniques Used: Inhibition, Expressing, Ex Vivo, One-tailed Test

    Effects of PMA/ionomycin stimulation on protein expression of human lamina propria (LP) immune cells Heatmaps representing the median fold change (FC) of cytokine and surface marker expression in CD45 + CD3 + T cells (top row) and CD45 + CD3 − immune cells (bottom row) isolated from colon lamina propria of five patients with Crohn's disease. Cells were treated with 20 ng/ml PMA and 1 μg/m (P/I) or DMSO (unstimulated) for 4 h in vitro , and protein expression was measured by mass cytometry (CyTOF). FC values are relative to unstimulated samples. Source data are available online for this figure.
    Figure Legend Snippet: Effects of PMA/ionomycin stimulation on protein expression of human lamina propria (LP) immune cells Heatmaps representing the median fold change (FC) of cytokine and surface marker expression in CD45 + CD3 + T cells (top row) and CD45 + CD3 − immune cells (bottom row) isolated from colon lamina propria of five patients with Crohn's disease. Cells were treated with 20 ng/ml PMA and 1 μg/m (P/I) or DMSO (unstimulated) for 4 h in vitro , and protein expression was measured by mass cytometry (CyTOF). FC values are relative to unstimulated samples. Source data are available online for this figure.

    Techniques Used: Expressing, Marker, Isolation, In Vitro, Mass Cytometry

    Global mass cytometric analysis of the immune cell composition in the colonic lamina propria of therapy refractory IBD patients Experimental setup for mass cytometric assays. Non‐inflamed: n = 4, CD: n = 6, UC: n = 6. FlowSOM plot of merged FCS files from LPMCs of non‐inflamed controls, UC or CD patients after stimulation with PMA/ionomycin ± 1 μM BTP2 for 4 h ex vivo . Colors of the t‐SNE plot represent 20 clusters of distinct CD45 + LPMC lineages. The heatmap shows the expression levels of 21 markers used for defining cell clusters. FlowSOM map of merged FCS files from CD45 + cells of non‐inflamed controls, UC and CD patients that were left unstimulated or treated with PMA/ionomycin ± 1 μM BTP2. Colors indicate altered cell clusters in CD patients vs. controls, UC patients vs. controls and CD vs. UC patients. viSNE plots of one exemplary non‐inflamed control, UC and CD patient colored by marker expression levels (blue: low, red: high). Quantified frequencies (%) of each cell subset defined by the cluster analysis. Statistical significance was calculated using the edgeR statistical framework with negative binomial GLM and a false discovery rate adjusted to 10% using the Benjiamini–Hochberg procedure (* P adj
    Figure Legend Snippet: Global mass cytometric analysis of the immune cell composition in the colonic lamina propria of therapy refractory IBD patients Experimental setup for mass cytometric assays. Non‐inflamed: n = 4, CD: n = 6, UC: n = 6. FlowSOM plot of merged FCS files from LPMCs of non‐inflamed controls, UC or CD patients after stimulation with PMA/ionomycin ± 1 μM BTP2 for 4 h ex vivo . Colors of the t‐SNE plot represent 20 clusters of distinct CD45 + LPMC lineages. The heatmap shows the expression levels of 21 markers used for defining cell clusters. FlowSOM map of merged FCS files from CD45 + cells of non‐inflamed controls, UC and CD patients that were left unstimulated or treated with PMA/ionomycin ± 1 μM BTP2. Colors indicate altered cell clusters in CD patients vs. controls, UC patients vs. controls and CD vs. UC patients. viSNE plots of one exemplary non‐inflamed control, UC and CD patient colored by marker expression levels (blue: low, red: high). Quantified frequencies (%) of each cell subset defined by the cluster analysis. Statistical significance was calculated using the edgeR statistical framework with negative binomial GLM and a false discovery rate adjusted to 10% using the Benjiamini–Hochberg procedure (* P adj

    Techniques Used: Ex Vivo, Expressing, Marker

    17) Product Images from "Type 17 Follicular Helper T (Tfh17) Cells are Superior for Memory Maintenance"

    Article Title: Type 17 Follicular Helper T (Tfh17) Cells are Superior for Memory Maintenance

    Journal: bioRxiv

    doi: 10.1101/2022.07.31.502219

    Human Tfh CM cells are enriched with the Tfh17 subset whereas Tfh EM cells are enriched with the Tfh1 subset (A-C) Human PBMC samples from 33 healthy blood donors were analyzed. Representative FACS plots and statistics showing the percentages of Tfh1, Tfh2 and Tfh17 cells in Tfh CM (A) or Tfh EM (B) subsets. Tfh CM/ Tfh EM ratios for Tfh1/2/17 in each individual were calculated (C). (D-E) FACS-purified Tfh EM and Tfh CM from 5 healthy individuals were analyzed for the expressions of indicated transcription factors by qPCR. The statistics for relative gene expression 2 -ΔΔCt (normalized to Tfh EM ) (D) and Tfh CM /Tfh EM ratios (E). (F-G) PBMC from 13 healthy individuals were analyzed for the secretions for indicated cytokines post PMA/ionomycin stimulation. The statistics for the percentages of cytokine + cells (F) and the Tfh CM /Tfh EM ratios (G). FC: average fold change. The p values were calculated by Friedman test.
    Figure Legend Snippet: Human Tfh CM cells are enriched with the Tfh17 subset whereas Tfh EM cells are enriched with the Tfh1 subset (A-C) Human PBMC samples from 33 healthy blood donors were analyzed. Representative FACS plots and statistics showing the percentages of Tfh1, Tfh2 and Tfh17 cells in Tfh CM (A) or Tfh EM (B) subsets. Tfh CM/ Tfh EM ratios for Tfh1/2/17 in each individual were calculated (C). (D-E) FACS-purified Tfh EM and Tfh CM from 5 healthy individuals were analyzed for the expressions of indicated transcription factors by qPCR. The statistics for relative gene expression 2 -ΔΔCt (normalized to Tfh EM ) (D) and Tfh CM /Tfh EM ratios (E). (F-G) PBMC from 13 healthy individuals were analyzed for the secretions for indicated cytokines post PMA/ionomycin stimulation. The statistics for the percentages of cytokine + cells (F) and the Tfh CM /Tfh EM ratios (G). FC: average fold change. The p values were calculated by Friedman test.

    Techniques Used: FACS, Purification, Real-time Polymerase Chain Reaction, Expressing

    18) Product Images from "Myeloid mechano-metabolic programming restricts anti-tumor immunity"

    Article Title: Myeloid mechano-metabolic programming restricts anti-tumor immunity

    Journal: bioRxiv

    doi: 10.1101/2022.07.14.499764

    a. Heat map of relative metabolite levels of CD3/CD28-activated CD8 + CTLs cultured for 24 h in medium containing a molar ratio of 1:1 [0.5 mM], 3:1 [1.5 mM], and 9:1 [4.5 mM] ornithine:arginine, LC-MS analysis. (n=3 biological replicates) b. Representative immunofluorescence microscopy of OVA-PyMT tumor cells challenged with GFP + -OTI CTLs (green) for 24 h in medium containing a molar ratio of 1:1 [0.5mM] or 3:1 [1.5 mM] ornithine:arginine, cleaved-caspase 3 (red) and DNA (blue), (Scale Bar: 40 µm). c. Graphical description of the experimental setup for d-j. d. Relative serum metabolites derived from retro-orbital isolated blood from mice containing PyMT tumors after 3 weeks of growth in C57BL6/J mice gavaged daily with 2 g/kg glycine, ornithine, arginine, or water (100 µL), 4 h prior to isolation of blood/serum, measured with LC-MS (n=8 pooled into two technical replicates). e. qPCR based assessment of circulating tumor cells via PyMT expression in blood clot isolated from blood collected for serum metabolite analysis (e), qPCR-ΔΔCT (housekeeping gene: 18s), (n=8). f. Percentage of PD1 + endogenous CD8 + CTLs isolated from stiff collagen PyMT tumors after 3 weeks of growth in C57BL6/J mice gavaged daily with 2 g/kg glycine, ornithine, arginine, or water (100 µL), (n=8). g. Percentage of CD44 + endogenous CD8 + CTLs isolated from stiff collagen PyMT tumors after 3 weeks of growth in C57BL6/J mice gavaged daily with 2 g/kg glycine, ornithine, arginine, or water (100 µL), (n=8). h-j Quantification of TCF and TOX in endogenous CD8 + CTLs isolated from stiff collagen PyMT tumors after 3 weeks of growth in C57BL6/J mice gavaged daily with 2 g/kg glycine, ornithine, arginine, or water (100 µL) and stimulated ex vivo with PMA [50 ng/mL] and ionomycin [500 ng/mL], (n=8). Data shown represent ± SEM. *P
    Figure Legend Snippet: a. Heat map of relative metabolite levels of CD3/CD28-activated CD8 + CTLs cultured for 24 h in medium containing a molar ratio of 1:1 [0.5 mM], 3:1 [1.5 mM], and 9:1 [4.5 mM] ornithine:arginine, LC-MS analysis. (n=3 biological replicates) b. Representative immunofluorescence microscopy of OVA-PyMT tumor cells challenged with GFP + -OTI CTLs (green) for 24 h in medium containing a molar ratio of 1:1 [0.5mM] or 3:1 [1.5 mM] ornithine:arginine, cleaved-caspase 3 (red) and DNA (blue), (Scale Bar: 40 µm). c. Graphical description of the experimental setup for d-j. d. Relative serum metabolites derived from retro-orbital isolated blood from mice containing PyMT tumors after 3 weeks of growth in C57BL6/J mice gavaged daily with 2 g/kg glycine, ornithine, arginine, or water (100 µL), 4 h prior to isolation of blood/serum, measured with LC-MS (n=8 pooled into two technical replicates). e. qPCR based assessment of circulating tumor cells via PyMT expression in blood clot isolated from blood collected for serum metabolite analysis (e), qPCR-ΔΔCT (housekeeping gene: 18s), (n=8). f. Percentage of PD1 + endogenous CD8 + CTLs isolated from stiff collagen PyMT tumors after 3 weeks of growth in C57BL6/J mice gavaged daily with 2 g/kg glycine, ornithine, arginine, or water (100 µL), (n=8). g. Percentage of CD44 + endogenous CD8 + CTLs isolated from stiff collagen PyMT tumors after 3 weeks of growth in C57BL6/J mice gavaged daily with 2 g/kg glycine, ornithine, arginine, or water (100 µL), (n=8). h-j Quantification of TCF and TOX in endogenous CD8 + CTLs isolated from stiff collagen PyMT tumors after 3 weeks of growth in C57BL6/J mice gavaged daily with 2 g/kg glycine, ornithine, arginine, or water (100 µL) and stimulated ex vivo with PMA [50 ng/mL] and ionomycin [500 ng/mL], (n=8). Data shown represent ± SEM. *P

    Techniques Used: Cell Culture, Liquid Chromatography with Mass Spectroscopy, Immunofluorescence, Microscopy, Derivative Assay, Isolation, Mouse Assay, Real-time Polymerase Chain Reaction, Expressing, Ex Vivo

    a. Heat map of relative metabolite levels of CD3/CD28-activated CD8 + CTLs cultured for 72 h in medium containing a molar ratio of 3:1 ornithine:arginine, medium refreshed every 24 h, LC-MS analysis. (n=3 biological replicates) b. Representative immunofluorescence microscopy of stiff-collagen OVA-PyMT tumor organoids challenged with GFP + -OTI CTLs (green) for 24 h in medium containing a molar ratio of 1:1 [0.5mM] or 3:1 [1.5 mM] ornithine:arginine, cleaved-caspase 3 (red) and DNA (blue), (Scale Bar: 40 µm). c. Graphical description of the experimental setup for d-f. d. Mean fluorescent intensity of PD1 staining of endogenous CD8 + CTLs isolated from stiff collagen PyMT tumors after 3 weeks of growth in C57BL6/J mice gavaged daily with 2 g/kg glycine, ornithine, arginine, or water (100 µL), (n=8). e. Mean fluorescent intensity of CD69 staining of endogenous CD8 + CTLs isolated from stiff collagen PyMT tumors after 3 weeks of growth in C57BL6/J mice gavaged daily with 2 g/kg glycine, ornithine, arginine, or water (100 µL), (n=8). f. Quantification of KI67 incorporation into endogenous CD8 + CTLs isolated from stiff collagen PyMT tumors after 3 weeks of growth in C57BL6/J mice gavaged daily with 2 g/kg glycine, ornithine, arginine, or water (100 µL) and stimulated ex vivo with PMA [50 ng/mL] and ionomycin [500 ng/mL], (n=8). g. Graphical description of the experimental setup for h-i. h. Tumor volume measurements of stiff collagen PyMT tumors over 4 weeks of growth in C57BL6/J mice gavaged daily with 2 g/kg glycine, ornithine, or arginine (100 µL) for the first 14 days and treated with isotype control or anti-PD1 blocking antibodies on day 5, 9, and 13, humane endpoint indicated by horizontal dotted line and white background (n=8) i. Kaplan-Meier survival curves of stage of experiment depicted in h. Mean survival of glycine treated isotype control and ornithine treated anti-PD1: 21 days, Mean survival of glycine and arginine treated anti-PD1: 28 days. Data shown represent ± SEM. *P
    Figure Legend Snippet: a. Heat map of relative metabolite levels of CD3/CD28-activated CD8 + CTLs cultured for 72 h in medium containing a molar ratio of 3:1 ornithine:arginine, medium refreshed every 24 h, LC-MS analysis. (n=3 biological replicates) b. Representative immunofluorescence microscopy of stiff-collagen OVA-PyMT tumor organoids challenged with GFP + -OTI CTLs (green) for 24 h in medium containing a molar ratio of 1:1 [0.5mM] or 3:1 [1.5 mM] ornithine:arginine, cleaved-caspase 3 (red) and DNA (blue), (Scale Bar: 40 µm). c. Graphical description of the experimental setup for d-f. d. Mean fluorescent intensity of PD1 staining of endogenous CD8 + CTLs isolated from stiff collagen PyMT tumors after 3 weeks of growth in C57BL6/J mice gavaged daily with 2 g/kg glycine, ornithine, arginine, or water (100 µL), (n=8). e. Mean fluorescent intensity of CD69 staining of endogenous CD8 + CTLs isolated from stiff collagen PyMT tumors after 3 weeks of growth in C57BL6/J mice gavaged daily with 2 g/kg glycine, ornithine, arginine, or water (100 µL), (n=8). f. Quantification of KI67 incorporation into endogenous CD8 + CTLs isolated from stiff collagen PyMT tumors after 3 weeks of growth in C57BL6/J mice gavaged daily with 2 g/kg glycine, ornithine, arginine, or water (100 µL) and stimulated ex vivo with PMA [50 ng/mL] and ionomycin [500 ng/mL], (n=8). g. Graphical description of the experimental setup for h-i. h. Tumor volume measurements of stiff collagen PyMT tumors over 4 weeks of growth in C57BL6/J mice gavaged daily with 2 g/kg glycine, ornithine, or arginine (100 µL) for the first 14 days and treated with isotype control or anti-PD1 blocking antibodies on day 5, 9, and 13, humane endpoint indicated by horizontal dotted line and white background (n=8) i. Kaplan-Meier survival curves of stage of experiment depicted in h. Mean survival of glycine treated isotype control and ornithine treated anti-PD1: 21 days, Mean survival of glycine and arginine treated anti-PD1: 28 days. Data shown represent ± SEM. *P

    Techniques Used: Cell Culture, Liquid Chromatography with Mass Spectroscopy, Immunofluorescence, Microscopy, Staining, Isolation, Mouse Assay, Ex Vivo, Blocking Assay

    19) Product Images from "Acidic Cannabinoids Suppress Proinflammatory Cytokine Release by Blocking Store-operated Calcium Entry"

    Article Title: Acidic Cannabinoids Suppress Proinflammatory Cytokine Release by Blocking Store-operated Calcium Entry

    Journal: Function

    doi: 10.1093/function/zqac033

    CBGA attenuates IL-2 production in Jurkat T cells. Evaluation of IL-2 levels and cell viability following treatment with CBGA. (A) Jurkat T cells were pretreated with CBGA at doses ranging from 0.1 µ m to 100 µ m for 30 min then stimulated with 1 µ m ionomycin and 20 ng/mL PMA. Supernatants were harvested at 24 h post-treatment and IL-2 concentrations were measured by ELISA. All data points represent mean ± SEM of four independent runs ( N = 4), each run containing a triplicate per condition. * P
    Figure Legend Snippet: CBGA attenuates IL-2 production in Jurkat T cells. Evaluation of IL-2 levels and cell viability following treatment with CBGA. (A) Jurkat T cells were pretreated with CBGA at doses ranging from 0.1 µ m to 100 µ m for 30 min then stimulated with 1 µ m ionomycin and 20 ng/mL PMA. Supernatants were harvested at 24 h post-treatment and IL-2 concentrations were measured by ELISA. All data points represent mean ± SEM of four independent runs ( N = 4), each run containing a triplicate per condition. * P

    Techniques Used: Enzyme-linked Immunosorbent Assay

    CBGA inhibitory potency is decreased by increasing serum concentrations. Serum-dependent effects of CBGA on I CRAC and NFAT activity in the presence of 0%, 1%, and 10% FBS. (A) Time course of CRAC currents recorded in Jurkat T cell in whole-cell patch-clamp experiments. Bath solution was supplemented with 0%, 1%, or 10% FBS (0% serum data is the same as in Figure 4A ). To elicit the active calcium depletion from the ER, 50 µ m IP 3 in the recording pipette was utilized. The resulting CRAC currents were allowed to reach maximal activation prior to extracellular application of CBGA at various concentrations. Current amplitudes were measured at −120 mV for each voltage ramp and were normalized to the control at 120 s. n represents the number of cells patched for each condition. (B) Concentration–response effects of CBGA on NFAT activity in the presence of 0%, 1%, and 10% FBS. A 10- and 12-point concentration-response curves, ranging from 100 n m to 100 µ m and 100 n m to 300 µ m , respectively, were established. The 12-point d/r was used for 10% FBS condition due to the weak activity of CBGA in the ten-point concentration range under this condition. Values are average ± SEM of 12–15 replicates, normalized to the control (cells treated with PMA + Ionomycin only).
    Figure Legend Snippet: CBGA inhibitory potency is decreased by increasing serum concentrations. Serum-dependent effects of CBGA on I CRAC and NFAT activity in the presence of 0%, 1%, and 10% FBS. (A) Time course of CRAC currents recorded in Jurkat T cell in whole-cell patch-clamp experiments. Bath solution was supplemented with 0%, 1%, or 10% FBS (0% serum data is the same as in Figure 4A ). To elicit the active calcium depletion from the ER, 50 µ m IP 3 in the recording pipette was utilized. The resulting CRAC currents were allowed to reach maximal activation prior to extracellular application of CBGA at various concentrations. Current amplitudes were measured at −120 mV for each voltage ramp and were normalized to the control at 120 s. n represents the number of cells patched for each condition. (B) Concentration–response effects of CBGA on NFAT activity in the presence of 0%, 1%, and 10% FBS. A 10- and 12-point concentration-response curves, ranging from 100 n m to 100 µ m and 100 n m to 300 µ m , respectively, were established. The 12-point d/r was used for 10% FBS condition due to the weak activity of CBGA in the ten-point concentration range under this condition. Values are average ± SEM of 12–15 replicates, normalized to the control (cells treated with PMA + Ionomycin only).

    Techniques Used: Activity Assay, Patch Clamp, Transferring, Activation Assay, Concentration Assay

    Effect of cannabinoids on NFAT activity in standard culture medium. Pure cannabinoids were tested at 5 µ m against NFAT activity. Jurkat T cells expressing NFAT reporter gene were pretreated with the cannabinoids before stimulation using 1 µ m ionomycin and 20 ng/mL PMA. Bioluminescence emitted by NFAT-mediated luciferase expression was measured in arbitrary units 5 h poststimulation. All data points represent mean ± SEM of 3 independent runs ( N = 3), with each run including n = 3 replicates per condition. * P
    Figure Legend Snippet: Effect of cannabinoids on NFAT activity in standard culture medium. Pure cannabinoids were tested at 5 µ m against NFAT activity. Jurkat T cells expressing NFAT reporter gene were pretreated with the cannabinoids before stimulation using 1 µ m ionomycin and 20 ng/mL PMA. Bioluminescence emitted by NFAT-mediated luciferase expression was measured in arbitrary units 5 h poststimulation. All data points represent mean ± SEM of 3 independent runs ( N = 3), with each run including n = 3 replicates per condition. * P

    Techniques Used: Activity Assay, Expressing, Luciferase

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    Thermo Fisher ionomycin
    Functional relevance of residues during activation. a Conformation of α3 around the hinge residue Gly 473 (circle) in structures obtained for the mutant F518H. b Lipid transport activity of Gly 473 mutants quantified in a cellular scrambling assay. Data shows fluorescence levels normalized to WT, at elevated Ca 2+ , 600 s after application of <t>ionomycin.</t> Dashed line indicates mean value of WT. Bars show mean of six biological replicates (depicted as spheres), errors are s.e.m. c Current magnitudes of HEK293T cells expressing WT or G473P recorded in excised patches. Bars represent mean of individual experiments ( n = 8), errors are s.e.m. d Surface expression of G473P. Anti-myc Western blot corresponds to biotinylated constructs pulled-down from HEK293T cells expressing myc-tagged WT or G473P after surface biotinylation. Samples are derived from the same experiment, gels and blots were processed in parallel. e Interactions between residues on α-helices 4 and 6 that are in contact in the active but not the inactive state of TMEM16F. f Coupling energies between residues depicted in e . Inset shows scheme of the double-mutant cycle analysis. WT/F518A/Q623A and WT/F518A/W619A refer to the cycle quantifying the effect of mutations F518A and either Q623A or W619A relative to WT, F518H/F518A/F518H_Q623A to the cycle of F518A and F518H_Q623A relative to the mutant F518H. g Contact region between α-helices 4 and 6 in the activated state of the double-mutant F518A_Q623A Ca . Blow up shows residues that are in contact as space-filling models. h Comparison of the open conformation of the active subunits of N562A Ca and F518A_Q623A Ca . Pore-lining helices are shown as Cα-trace.
    Ionomycin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher phorbol myristate acetate pma ionomycin
    Functional relevance of residues during activation. a Conformation of α3 around the hinge residue Gly 473 (circle) in structures obtained for the mutant F518H. b Lipid transport activity of Gly 473 mutants quantified in a cellular scrambling assay. Data shows fluorescence levels normalized to WT, at elevated Ca 2+ , 600 s after application of <t>ionomycin.</t> Dashed line indicates mean value of WT. Bars show mean of six biological replicates (depicted as spheres), errors are s.e.m. c Current magnitudes of HEK293T cells expressing WT or G473P recorded in excised patches. Bars represent mean of individual experiments ( n = 8), errors are s.e.m. d Surface expression of G473P. Anti-myc Western blot corresponds to biotinylated constructs pulled-down from HEK293T cells expressing myc-tagged WT or G473P after surface biotinylation. Samples are derived from the same experiment, gels and blots were processed in parallel. e Interactions between residues on α-helices 4 and 6 that are in contact in the active but not the inactive state of TMEM16F. f Coupling energies between residues depicted in e . Inset shows scheme of the double-mutant cycle analysis. WT/F518A/Q623A and WT/F518A/W619A refer to the cycle quantifying the effect of mutations F518A and either Q623A or W619A relative to WT, F518H/F518A/F518H_Q623A to the cycle of F518A and F518H_Q623A relative to the mutant F518H. g Contact region between α-helices 4 and 6 in the activated state of the double-mutant F518A_Q623A Ca . Blow up shows residues that are in contact as space-filling models. h Comparison of the open conformation of the active subunits of N562A Ca and F518A_Q623A Ca . Pore-lining helices are shown as Cα-trace.
    Phorbol Myristate Acetate Pma Ionomycin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Functional relevance of residues during activation. a Conformation of α3 around the hinge residue Gly 473 (circle) in structures obtained for the mutant F518H. b Lipid transport activity of Gly 473 mutants quantified in a cellular scrambling assay. Data shows fluorescence levels normalized to WT, at elevated Ca 2+ , 600 s after application of ionomycin. Dashed line indicates mean value of WT. Bars show mean of six biological replicates (depicted as spheres), errors are s.e.m. c Current magnitudes of HEK293T cells expressing WT or G473P recorded in excised patches. Bars represent mean of individual experiments ( n = 8), errors are s.e.m. d Surface expression of G473P. Anti-myc Western blot corresponds to biotinylated constructs pulled-down from HEK293T cells expressing myc-tagged WT or G473P after surface biotinylation. Samples are derived from the same experiment, gels and blots were processed in parallel. e Interactions between residues on α-helices 4 and 6 that are in contact in the active but not the inactive state of TMEM16F. f Coupling energies between residues depicted in e . Inset shows scheme of the double-mutant cycle analysis. WT/F518A/Q623A and WT/F518A/W619A refer to the cycle quantifying the effect of mutations F518A and either Q623A or W619A relative to WT, F518H/F518A/F518H_Q623A to the cycle of F518A and F518H_Q623A relative to the mutant F518H. g Contact region between α-helices 4 and 6 in the activated state of the double-mutant F518A_Q623A Ca . Blow up shows residues that are in contact as space-filling models. h Comparison of the open conformation of the active subunits of N562A Ca and F518A_Q623A Ca . Pore-lining helices are shown as Cα-trace.

    Journal: Nature Communications

    Article Title: Structural basis for the activation of the lipid scramblase TMEM16F

    doi: 10.1038/s41467-022-34497-x

    Figure Lengend Snippet: Functional relevance of residues during activation. a Conformation of α3 around the hinge residue Gly 473 (circle) in structures obtained for the mutant F518H. b Lipid transport activity of Gly 473 mutants quantified in a cellular scrambling assay. Data shows fluorescence levels normalized to WT, at elevated Ca 2+ , 600 s after application of ionomycin. Dashed line indicates mean value of WT. Bars show mean of six biological replicates (depicted as spheres), errors are s.e.m. c Current magnitudes of HEK293T cells expressing WT or G473P recorded in excised patches. Bars represent mean of individual experiments ( n = 8), errors are s.e.m. d Surface expression of G473P. Anti-myc Western blot corresponds to biotinylated constructs pulled-down from HEK293T cells expressing myc-tagged WT or G473P after surface biotinylation. Samples are derived from the same experiment, gels and blots were processed in parallel. e Interactions between residues on α-helices 4 and 6 that are in contact in the active but not the inactive state of TMEM16F. f Coupling energies between residues depicted in e . Inset shows scheme of the double-mutant cycle analysis. WT/F518A/Q623A and WT/F518A/W619A refer to the cycle quantifying the effect of mutations F518A and either Q623A or W619A relative to WT, F518H/F518A/F518H_Q623A to the cycle of F518A and F518H_Q623A relative to the mutant F518H. g Contact region between α-helices 4 and 6 in the activated state of the double-mutant F518A_Q623A Ca . Blow up shows residues that are in contact as space-filling models. h Comparison of the open conformation of the active subunits of N562A Ca and F518A_Q623A Ca . Pore-lining helices are shown as Cα-trace.

    Article Snippet: Images were acquired every 10 s. After 3 min, 50 µl of Buffer B containing 10 mM HEPES pH 7.4, 155 mM NaNO3 , 2.5 mM CaCl2 , 5% annexin V Alexa Fluor 594 conjugate, 5 nM Sytox red, and 100 µM ionomycin (Thermo Fischer) were added rapidly and cells were imaged for another 10 min. Data was evaluated in FIJI .

    Techniques: Functional Assay, Activation Assay, Mutagenesis, Activity Assay, Fluorescence, Expressing, Western Blot, Construct, Derivative Assay

    Characterization of activating mutants. a Ca 2+ -concentration-response relationships of mutants of Phe 518 measured in inside-out patches. b EC 50 values of Ca 2+ plotted against the normalized hydrophobicity (Eisenberg and Weiss Scale) of the respective Phe 518 mutations. Line shows a fit to the data. c Ca 2+ -concentration-response relationships of mutants of Asn 562 and d Tyr 563 measured in inside-out patches. a , c , d Data show averages of multiple experiments derived from independent cells (WT n = 10, F518H n = 5, F518Q n = 4, F518Y n = 5-6, F518A n = 5-9, F518I n = 7, N562A, n = 5, N562L n = 4, Y563A n = 5, Y563H n = 3–6), errors are s.e.m., lines show fit to a Hill equation. e , f Lipid transport of Phe 518 mutants quantified in a cellular scrambling assay. e Initial values recorded at resting Ca 2+ concentration and f levels measured 600 s after application of ionomycin, which increases intracellular Ca 2+ . Individual experiments are depicted as spheres (mock n = 10, F518I, F518A, F518Y n = 3, F518Q n = 4, F518H n = 6), errors are s.e.m. g , h Liposome-based in vitro scrambling assay of the reconstituted mutant F518H in comparison to WT. g Time-dependent fluorescence decrease upon addition of the reducing agent dithionite (t = 60 s) at 0 and 1000 µM Ca 2+ compared to WT. Data show mean of three technical replicates, errors (s.e.m.) are smaller than the displayed line width. h Ca 2+ -concentration response relationship of scrambling of the mutant F518H compared to WT obtained from three technical replicates (displayed in Supp. Fig. 2d ). Values were obtained as described in the methods. Solid line shows fit to a Hill equation, errors are s.e.m. Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: Structural basis for the activation of the lipid scramblase TMEM16F

    doi: 10.1038/s41467-022-34497-x

    Figure Lengend Snippet: Characterization of activating mutants. a Ca 2+ -concentration-response relationships of mutants of Phe 518 measured in inside-out patches. b EC 50 values of Ca 2+ plotted against the normalized hydrophobicity (Eisenberg and Weiss Scale) of the respective Phe 518 mutations. Line shows a fit to the data. c Ca 2+ -concentration-response relationships of mutants of Asn 562 and d Tyr 563 measured in inside-out patches. a , c , d Data show averages of multiple experiments derived from independent cells (WT n = 10, F518H n = 5, F518Q n = 4, F518Y n = 5-6, F518A n = 5-9, F518I n = 7, N562A, n = 5, N562L n = 4, Y563A n = 5, Y563H n = 3–6), errors are s.e.m., lines show fit to a Hill equation. e , f Lipid transport of Phe 518 mutants quantified in a cellular scrambling assay. e Initial values recorded at resting Ca 2+ concentration and f levels measured 600 s after application of ionomycin, which increases intracellular Ca 2+ . Individual experiments are depicted as spheres (mock n = 10, F518I, F518A, F518Y n = 3, F518Q n = 4, F518H n = 6), errors are s.e.m. g , h Liposome-based in vitro scrambling assay of the reconstituted mutant F518H in comparison to WT. g Time-dependent fluorescence decrease upon addition of the reducing agent dithionite (t = 60 s) at 0 and 1000 µM Ca 2+ compared to WT. Data show mean of three technical replicates, errors (s.e.m.) are smaller than the displayed line width. h Ca 2+ -concentration response relationship of scrambling of the mutant F518H compared to WT obtained from three technical replicates (displayed in Supp. Fig. 2d ). Values were obtained as described in the methods. Solid line shows fit to a Hill equation, errors are s.e.m. Source data are provided as a Source Data file.

    Article Snippet: Images were acquired every 10 s. After 3 min, 50 µl of Buffer B containing 10 mM HEPES pH 7.4, 155 mM NaNO3 , 2.5 mM CaCl2 , 5% annexin V Alexa Fluor 594 conjugate, 5 nM Sytox red, and 100 µM ionomycin (Thermo Fischer) were added rapidly and cells were imaged for another 10 min. Data was evaluated in FIJI .

    Techniques: Concentration Assay, Derivative Assay, In Vitro, Mutagenesis, Fluorescence