W1 spinning disk unit

For immunocytochemistry of naïve CD8 T cells in suspension, cells were fixed with 4% paraformaldehyde (PFA) in phosphate buffer for 15 min, washed with PBS, and allowed to settle for 45 min onto poly-l-lysine (P6282, Sigma)–coated coverslips (Matsunami) at 37°C. Cells were then washed with PBS, permeabilized with 0.3% Triton-X100 in PBS for 10 min, and blocked with 1% bovine serum albumin in PBS for 45 min at room temperature. The cells were then incubated with indicated primary antibodies at 4°C overnight, washed, and stained with secondary antibodies for 45 min at room temperature. For F-actin staining, cells were incubated with fluorescent dye–labeled phalloidin diluted in 0.3% Triton-X100/PBS for 20 min at room temperature. Cells were finally washed with PBS and mounted onto slides with ProLong Diamond Antifade reagent (P36970, Life Technologies). Fluorescence images were acquired with an SD-OSR IX83 inverted microscope (Olympus) equipped with a 100× numerical aperture (NA) 1.4, UPLSAPO oil immersion objective (Olympus) and Yokogawa W1 spinning disk unit (Yokogawa) controlled by MetaMorph software (Universal Imaging). Quantification of F-actin, pZap70 [Y319], and pLAT [Y171] staining intensity was performed using ImageJ software. Coefficient of variant of F-actin staining intensity was calculated as the ratio of the SD and average staining intensity.