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. 2024 Sep 13;15:8008. doi: 10.1038/s41467-024-52024-y

Fig. 4. Ca2+ microdomains evoked by MASTER-NAADP in different cell types.

Fig. 4

Representative high-resolution Ca2+ images of Jurkat T cells loaded with both Fluo4-AM and Fura-Red-AM after stimulation with 100 nM MASTER-NAADP (A) or 100 nM MASTER-NADP (B). Number of cells indicated in figure. A, B (upper panel): Pseudocolor images indicate emission ratios between Fluo-4 and Fura-Red ranging from 0.3 -1.3; ratio data were then converted using external calibration corresponding to 8 to 182 nM [Ca2+]i. Scale bars, 5 µm for whole cells. A, B (lower panel): Magnified regions as indicated as 3D surface plots. C Analysis across the first 15 s of stimulation shown as number of Ca2+ microdomains. C Data are displayed as mean ± SEM; Jurkat WT MASTER-NAADP, n = 66 cells; Jurkat WT MASTER-NADP, n = 40 cells; Jurkat Hn1l/Jpt2-/- MASTER-NAADP, n = 43 cells; Jurkat Hn1l/Jpt2-/- MASTER-NADP, n = 37 cells; Jurkat WT DMSO, n = 17 cells; nonparametric Kruskal-Wallis test and Dunn’s correction for multiple testing *P < 0.05; **P < 0.005. D (upper panel) Representative high-resolution Ca2+ images of primary murine T cells loaded with both Fluo4-AM and Fura-Red-AM after stimulation with 100 nM MASTER-NAADP. Pseudocolor images indicate emission ratios between Fluo-4 and Fura-Red ranging from 0.3–1.3; ratio data were then converted using external calibration corresponding to 0 to 347 nM [Ca2+]i. Scale bars, 5 µm for whole cells. D (lower panel) Magnified regions as indicated as 3D surface plots. E Analysis across the first 15 s of stimulation shown as number of Ca2+ microdomains. E Data are displayed as mean ± SEM; MASTER-NAADP, n = 89 cells; MASTER-NADP, n = 58 cells; two-sided, nonparametric Mann-Whitney U test: **P < 0.005. F (upper panel) Representative high-resolution Ca2+ images of human NK cells (KHYG-1) loaded with both Fluo4-AM and Fura-Red-AM after stimulation with 100 µM MASTER-NAADP. Pseudocolor images indicate emission ratios between Fluo-4 and Fura-Red ranging from 0.3–2.0; ratio data were then converted using external calibration corresponding to 5 to 293 nM [Ca2+]i. Scale bars, 5 µm for whole cells. F (lower panel) Magnified regions as indicated as 3D surface plots. G Analysis across the first 15 s of stimulation shown as number of Ca2+ microdomains. G Data are displayed as mean ± SEM; MASTER-NAADP, n = 46 cells; MASTER-NADP, n = 39 cells; two-sided, nonparametric Mann-Whitney U test: *P < 0.05. H (upper panel) Representative high-resolution Ca2+ images of Neuro2A cells loaded with both Fluo4-AM and Fura-Red-AM after stimulation with 100 µM MASTER-NAADP. H (lower panel) Magnified regions as indicated as 3D surface plots. Pseudocolor images indicate emission ratios between Fluo-4 and Fura-Red ranging from 0.3–1.3; ratio data were then converted using external calibration corresponding to 0 to 147 nM [Ca2+]i. I Analysis across the first 15 s of stimulation shown as number of Ca2+ microdomains. I Data are mean ± SEM; Neuro2A WT MASTER-NAADP, n = 20 cells; Neuro2A WT MASTER-NADP, n = 23 cells; Neuro2A Hn1l/Jpt2-/- MASTER-NAADP, n = 21 cells; Neuro2A Hn1l/Jpt2-/- MASTER-NADP, n = 20 cells; nonparametric Kruskal-Wallis test and Dunn’s correction for multiple testing *P < 0.05; **P < 0.005; ***P < 0.001. Source data for (C, E, G, I) and exact p values are provided as a Source Data file.