Fig 1: Low extracellular Ca2+ condition induces rapid activation of integrin αLβ2.Mouse splenic T cells were suspended in a buffer containing 0.6 mM Mg2+ plus the indicated concentration of Ca2+. 0 mM [Ca2+] means a Ca2+ concentration below the detection limit (2.34 μM Ca2+) of the Calcium Quantitation Kit (36361, AAT Bioquest). a ICAM-1 (20 μg/ml) was immobilized on petri dishes. Adhesion of T cells to the immobilized ICAM-1 substrates at a wall shear stress of 1 dyn/cm2 was examined (n = 6). b Binding of soluble ICAM-1 to T cells was calculated with the specific mean fluorescence intensity (MFI) (n = 6). c Schematic diagrams of the experimental setup for integrin αLβ2 tail FRET system (αLCloverβ2mRuby2) to monitor the separation of αLβ2 cytoplasmic domains (bottom) and the strategy to generate Itgal-LSL-Clover;Itgb2-LSL-mRuby2;CD4-Cre mice bearing T cells expressing αLCloverβ2mRuby2 (top). d Representative pseudocolored αLβ2 tail FRET ratio (FmRuby2/FClover) images of T cells expressing αLCloverβ2mRuby2 on the immobilized ICAM-1 (20 μg/ml) substrates. Scale bar, 6 µm. Images are from one representative experiment out of three. e Quantification of αLβ2 tail FRET ratio. The FRET ratio of each cell was normalized to the mean value of cells in 1.2 mM Ca2+. Data are presented as box-and-whisker plots showing the median (central line), 25th–75th percentile (bounds of the box), and 5th–95th percentile (whiskers) (n = 30 cells for each condition from 3 experiments). f Time course of αLβ2 tail FRET ratio change in T cells on the immobilized ICAM-1 (20 μg/ml) substrates upon chelation of Ca2+ with 5 mM EGTA in buffer containing 1.2 mM Ca2+ plus 0.6 mM Mg2+ (left). EGTA was added at time point 0. The FRET ratio change was normalized to the mean value of cells before EGTA treatment. The solid lines represent the mean; shaded areas, s.e.m. (n = 50 cells from 3 experiments). The statistic results at representative time points were shown (right). Data represent the mean ± s.e.m. in (a, b and f). ns, not significant (one-way ANOVA with Dunnett’s test in (a), Brown-Forsythe and Welch one-way ANOVA with Dunnett’s test in (b) and (e) to compare the means of different Ca2+ concentration groups to the mean of 1.2 mM Ca2+ group; one-way ANOVA with Dunnett’s test in (f) to compare the means of 15 s and 30 s groups to the mean of 0 s group). Source data are provided as a Source Data file.