fluorescent ca2+ indicator dye fluo-4 am  (Thermo Fisher)

Journal: Frontiers in Physiology

Article Title: Elucidation of the binding mode of organic polysulfides on the human TRPA1 receptor

doi: 10.3389/fphys.2023.1180896

Figure Lengend Snippet: DMTS dose–effect curves in TRPA1 mutant variants, compared to the effect of 1 mM carvacrol. Fluo-4 calcium-sensitive fluorescence was measured by flow cytometry in different TRPA1 variants, using increasing DMTS concentration, compared to the fluorescent signal of 1 mM carvacrol (100%). The single mutation of C621, C641, and C834 decreased the effect of DMTS in TRPA1, but C621A/C641A double mutant and C621A/C641A/C665A triple mutant showed insensitivity to DMTS, indicating that these amino acids are involved in the binding site of the organic polysulfides. The combined double mutation of C727 and C834 (C727A/C834A) made no significant difference from the wild-type TRPA1, suggesting that they are not parts of the binding site of the organic polysulfides. Mean ± SEM, N = 2-4 (number of measurements per TRPA1 variation), n > 10000 (number of cells per measurement).

Article Snippet: In total, 1 ml (∼100,000 cells) of each transfected CHO cell line was stained by 4 µl Invitrogen Fluo-4, AM (Invitrogen, Thermo Fisher Scientific F14201) cell permeant calcium indicator fluorescent dye at 37 °C for 1 h in non-adhesive tubes.

Techniques: Mutagenesis, Fluorescence, Flow Cytometry, Concentration Assay, Binding Assay

Journal: Frontiers in Physiology

Article Title: Elucidation of the binding mode of organic polysulfides on the human TRPA1 receptor

doi: 10.3389/fphys.2023.1180896

Figure Lengend Snippet: TRPA1 activity measured by calcium-sensitive fluorescence flow cytometry. The different TRPA1 variant-expressing CHO cells were stained by Fluo-4 calcium-sensitive fluorophore. TRPA1 activation causes calcium influx, and therefore increases the intensity of fluorescence, which was measured by flow cytometry. The signal intensity was compared to the effect of 100 µM carvacrol, which was used as a non-electrophilic control agonist. Relatively high concentration (100 µM) of organic polysulfides (DMTS, 90% DADS and 63.5% DATS) showed only slight changes in their receptor-activating effect in the case of TRPA1 single mutants (C621A, C641A, and C665A), but their effect was completely eliminated in the triple-mutant TRPA1 variant (C621A/C641A/C665A). The effect of JT010 was eliminated even in the C621A single mutant, and the other two single mutants also showed a tendency for decreased JT010 sensitivity. Diagram shows the means ± SEM, two-way ANOVA with Bonferroni’s multiple comparison test, significance compared to WT TRPA1: * p < 0.0001, WT TRPA1 replication experiments (N = 4-12), mutant TRPA1 replication experiments (N = 2-5), sample size ( n = 4–19), cell number per sample ( n > 2000).

Article Snippet: In total, 1 ml (∼100,000 cells) of each transfected CHO cell line was stained by 4 µl Invitrogen Fluo-4, AM (Invitrogen, Thermo Fisher Scientific F14201) cell permeant calcium indicator fluorescent dye at 37 °C for 1 h in non-adhesive tubes.

Techniques: Activity Assay, Fluorescence, Flow Cytometry, Variant Assay, Expressing, Staining, Activation Assay, Concentration Assay, Mutagenesis