The R65Q mutation does not affect the subcellular localization of STX11 or the number and polarization of perforin-containing granules. (A) CTLs from a control individual or P1 were incubated in the presence or absence of anti-CD3-coated P815 cells at a 1:1 ratio for 15 minutes at 37°C on polylysine-coated coverslips. Cells were fixed, permeabilized, and stained using mouse anti-perforin-1 antibody, rabbit anti-STX11 antibody and Alexa 633-Phalloidin. Larger arrowheads show the intracellular vesicular pool of STX11. Smaller arrows show the fraction of STX11 localizing at the plasma membrane. Bars represent 5 μm. Insets display a zoomed-in view of the selected area. (B) Pearson’s colocalization coeficient (PCC) between perforin 1 and STX11 in the set of images shown in panel A. Values represent the mean ± standard deviation (SD); n = 15 cells. Quantification of the number of perforin-containing granules per cell (C) and the number of cells containing perforin granules (D) was performed using stimulated emission depletion images (as shown in panel A; nonconjugated). Data represent the mean ± SD; n = 25 cells for panel C and n = 100 cells for panel D. (E) Quantification of the number of CTLs making contact with target cells that display polarized granules. Stimulated emission depletion images (as shown in panel A; conjugated) were used to determine the percentage of CTLs in contact with target cells that exhibited polarized perforin-containing granules at the immunologic synapse among the total number of CTLs containing perforin granules. Data represent the mean ± SD; n = 50 cells.