Fig. 3: Ionomycin induces [Ca²⁺]_ex drop and integrin αLβ2 activation on T cell surface.

Splenic T cells were isolated from R26-LSL-CEPIAexternal;Itgal-LSL-Clover;Itgb2-LSL-mRuby2;CD4-Cre mice and suspended in buffer containing 1.2 mM Ca²⁺ and 0.6 mM Mg²⁺. Ionomycin (final concentration 1 μM) was added at time point 0. a Representative pseudocolor image of CEPIAexternal ratio on the T cell surface in response to ionomycin stimulation. The first two images show the distribution of CEPIAexternal in Em450 and Em510 channels. Scale bar, 3 µm. Images are from one representative experiment out of three. b Time course of CEPIAexternal ratio change on the T cell surface in response to ionomycin stimulation (n = 30 cells from 3 experiments). c [Ca²⁺]_ex curve was plotted by converting the CEPIAexternal ratio in (b) to Ca²⁺ concentration according to the Ca² calibration curve in Fig. 2f (left), and [Ca²⁺]_ex values at the representative time points were shown (right) (n = 65 cells from 3 experiments). d Ca²⁺ concentration in the buffer was measured using a Calcium Colorimetric Assay Kit (S1063S, Beyotime) before and after ionomycin treatment (n = 6). e Time course of CEPIAexternal ratio change in T cells pretreated with 100 µM BAPTA-AM or DMSO vehicle control in response to ionomycin stimulation (left) and CEPIAexternal ratios at the representative time points were shown (right) (n = 60 cells from 3 experiments). f Representative pseudocolor images of the αLβ2 tail FRET ratio on the T cell surface in response to ionomycin stimulation. The first two images show the Clover and mRuby2 signals. Scale bar, 3 µm. Images are from one representative experiment out of three. g Time course of αLβ2 tail FRET ratio change in T cells pretreated with 100 µM BAPTA-AM or DMSO vehicle control in response to ionomycin stimulation (left), and the normalized FRET ratios at the representative time points were shown (right) (n = 60 cells from 3 experiments). The FRET ratio is normalized to the mean value of cells before the addition of stimuli (R/R₀). h Effect of ionomycin treatment on the adhesion of T cells pretreated with 100 µM BAPTA-AM or DMSO vehicle control to the immobilized ICAM-1 (20 μg/ml) substrates at a wall shear stress of 1 dyn/cm² (n = 3). The solid lines represent the mean; shaded areas, s.e.m. in (b, c, e, and g). Data represent the mean ± s.e.m. in (c, d, e, g and h). ns, not significant (unpaired two-tailed Student’s t test and unpaired two-tailed Welch’s unequal variance t test in (d, e, and g); one-way ANOVA with Dunnett’s test in (h). Source data are provided as a Source Data file.