Journal: Biomolecules

Article Title: TRPA1 Covalent Ligand JT010 Modifies T Lymphocyte Activation

doi: 10.3390/biom14060632

Figure Lengend Snippet: Analyses of concentration-dependent effects of JT010 alone on mice peritoneal (PEC) cells and thymocytes. ( a ) Gating strategy and representative flow cytometry plots of peritoneal cells stained by antibodies to CD4, CD8, B220, and CD14 combinations. Upper left panel shows G1–G4 gates based on FSC versus SSC dot plot of cells. Middle and bottom left panels show cells of G1 gate stained either anti-CD4 and CD8 antibodies or anti-CD4 and B220 antibodies. Right panels show results of anti-CD4 and anti-CD14 labeling of the cells in gates G4, G3, and G2, in order from top to bottom, respectively. Analysis gates surrounded by thick rectangles sign gates for CD4 + cells, B220 + cells, and CD14 + cells in the middle left panel, in the bottom left panel, and in the middle right panel, respectively. Graphs ( b – d ) show JT010, DMSO, or TcR (CD3)-stimulated time-dependent changes in intracellular Ca 2+ levels ( b ) in PEC lymphocytes (G1 gate), ( c ) PEC CD4 + lymphocytes, or ( d ) thymocytes. Isolated cells were surface-labeled with CD4 antibody and then loaded with Fluo-3-AM. Fluo-3 fluorescence that is proportional to the intracellular Ca 2+ levels was detected by flow cytometry. Mean fluorescence intensity (MFI) of non-stimulated cells was measured, then different concentrations of JT010, DMSO as vehicle control-induced changes, and TcR-activated Ca 2+ signals (CD3) were monitored. Graphs show fold of changes in free intracellular Ca 2+ -levels calculated as a ratio to that of quiescent cells. Data are presented as mean ± SEM, n = 3, * p < 0.05. ( e ) Phosphatidydylserine (PS) exposure of cells monitored by fluorescent Annexin V-binding measured by flow cytometry. Addition of 4, 8, 20, or 40 μM of JT010, vehicle control DMSO, and TcR-stimulated (CD3) time-dependent changes in mean fluorescent intensity (MFI) values over time of a representative experiment series are shown in PEC lymphocytes. At the end of each experiment, addition of ionomycin for internal control shows maximum response of PS exposure. Data are presented as mean ± SEM, b,c n = 4, d n = 3, e representative measurement, * p < 0.05.

Article Snippet: For TcR-dependent Ca 2+ signal analyses, cells were stimulated with hamster anti-mouse CD3 Epsilon monoclonal antibody (Clone 145-2C11, Cat. No.: MAB484, RD systems, Minneapolis, MN, USA) for 10 min, then crosslinked with goat anti-hamster IgG (Abcam, Cat. No.: ab5738) as described earlier [ , , ].

Techniques: Concentration Assay, Flow Cytometry, Staining, Labeling, Isolation, Fluorescence, Control, Binding Assay

Journal: Biomolecules

Article Title: TRPA1 Covalent Ligand JT010 Modifies T Lymphocyte Activation

doi: 10.3390/biom14060632

Figure Lengend Snippet: Effects of JT010 on TcR-induced calcium signals and on plasma membrane integrity in lymphocytes and in CD4 + lymphocytes isolated from peritoneal cavity of mice. ( a – d ) Isolated cells were labeled with anti-CD4 and anti-CD8 antibodies, then incubated with 1–40 μM of JT010 or vehicle control DMSO for 10 min before activation of cells via the T cell receptor (TcR)-induced by anti-CD3 antibody cross-linking. Changes in intracellular Ca 2+ levels in cells were assessed by monitoring the fluorescence of the Ca 2+ indicator Fluo-3 by flow cytometry. Lymphocytes ( a , c , e ) and CD4 + lymphocytes ( b , d ) were gated as detailed in a. ( a , b ) Graphs show TcR-stimulated time-dependent changes in intracellular Ca 2+ levels over time; ( c , d ) show JT010 concentration dependence of the maximal response measured at 240 s. Data are presented as mean ± SEM, n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001. ( e ) Effects of 40 μM JT010 on the plasma membrane integrity of lymphocytes measured by annexin V–binding assay. Changes in fluorescent annexin V–binding to cell surface (upper panel), and 7-AminoactinomycinD (7-AAD) labeling of the same cells (middle panel) were recorded by flow cytometry over time. Cell-surface-bound Annexin V fluorescence is proportional to the phosphatidylserine (PS) exposure of the cells and 7-AAD fluorescence is proportional to cell DNA staining in nuclei, both reflecting compromised plasma membrane integrity. At the end of the experiment, ionomycin was applied for internal control to check nonresponsive cells and acquire a maximum response of PS exposure. Bottom left panel shows Annexin V fluorescence plotted against 7-AAD fluorescence at the beginning of the measurement; bottom right panel shows the same after the addition of ionomycin. Representative flow cytometric plots demonstrate the results of n = 3 experiment.

Article Snippet: For TcR-dependent Ca 2+ signal analyses, cells were stimulated with hamster anti-mouse CD3 Epsilon monoclonal antibody (Clone 145-2C11, Cat. No.: MAB484, RD systems, Minneapolis, MN, USA) for 10 min, then crosslinked with goat anti-hamster IgG (Abcam, Cat. No.: ab5738) as described earlier [ , , ].

Techniques: Membrane, Isolation, Labeling, Incubation, Control, Activation Assay, Fluorescence, Flow Cytometry, Concentration Assay, Binding Assay, Staining