Reduction of negative charge at the inner leaflet of the T cell synapse during early stages of TCR engagement by peptide-MHC. (A and B) B-A8 T cells expressing the anionic lipid probe R-pre-mRFP were stimulated with HA-DR4/ICAM-1 lipid beads and imaged by confocal microscopy at 10-s intervals. Top row: typical cell showing bead (blue) and R-pre binding to inner leaflet (red) as synapse is formed. Bottom row shows intensity of R-pre fluorescence using pseudo-color gradient; quantification within or outside of synapse (B). Images representative of n > 3 experiments. Bar, 5 µm. (C and E) B-A8 T cells expressing both R-pre-mRFP and ZAP-70-eGFP probes (C) or MyrPalm-eGFP probe (E) were stimulated with HA-DR4/ICAM-1 lipid beads and imaged by confocal microscopy. Bottom rows show higher magnification (white box) and pseudo-color gradient of fluorescence intensity. Dotted line represents cross section for cells shown in D and F. Images representative of n > 3 experiments. Bar, 10 µm. (D and F) Fluorescence signals of bead (blue), R-pre (red), and ZAP-70 (green; D) or MyrPalm (green; F) across cells in C and E (dotted lines). (G) Population analysis of ratio between PM fluorescence within or outside bead–cell interface for at least 30 cells per group, error bars show SEM. P-value was calculated using an unpaired two-tailed Student’s t test with a 99% CI. n > 10 cells per experiment.