Changes in lipid composition of TCR microclusters occur even when Lck activity is inhibited. (A) B-A8 T cells expressing R-pre-mRFP and ZAP-70-eGFP probes were treated with 50 µM PP2 before and during stimulation with HA-DR4/ICAM-1 lipid beads. Cells were imaged by confocal microscopy (10-s intervals). Bottom row shows higher magnification of bead–cell interface (white box). Images representative of n > 3 experiments. Bar, 10 µm. (B) WT B-A8 T cells were labeled with calcium probe Fluo-4AM and incubated with or without ±50 µM PP2 treatment. Cells were then stimulated with HA-DR4/ICAM-1 lipid beads ±PP2. Calcium flux was imaged during bead stimulation (10-s intervals, 10 min) by wide-field microscopy. Typical cells for each condition are shown at three time points. (top row) Intensity of Fluo-4 signal in pseudo-color gradient. (bottom row) DIC images at same time points. Bars, 10 µm. (C) Fluo-4 intensities during bead stimulation ±PP2 treatment (n = 10 cells per time point). Data representative of n = 3 experiments. (D) Population analysis of relative R-pre fluorescence intensities at PM of bead–cell interface versus irrelevant membrane for cells stimulated with HA-DR4/ICAM-1 lipid beads ± treatment with 50 µM PP2 (n ≥ 30 cells). P-value was calculated with an unpaired two-tailed Student’s t test with a 99% CI (*, 0.0237). n = 10 cells per experiment.