Alexa fluor 488 or alexa fluor 568 phalloidin

Cells were grown on 0.001% poly-L-lysine-coated glass coverslips (Carolina Biological Supply, #633029). Cells were fixed for 15 min at 37°C in warm 4% paraformaldehyde (diluted from 16% in PBS, Electron Microscopy Sciences) plus 4% sucrose in PBS. Coverslips were blocked for 30 min in 10% goat serum (Thermo Fisher, 16210-072) plus 0.1% Triton X-100 (MilliporeSigma) in PBS and incubated overnight at 4°C with primary antibody diluted in 3% goat serum plus 0.1% Triton X-100 in PBS, and similarly incubated for 45 min at room temperature with secondary antibodies or phalloidin. Coverslips were mounted in Prolong Gold Antifade mounting media (Thermo Fisher). Primary antibodies, their usage, and source are given in Table 1. Secondary antibodies are as follows: goat anti-mouse/rabbit Alexa Fluor 405, 488, 568, or 647 (Thermo Fisher, 1:200), and goat anti-chicken Alexa Fluor 488 (Thermo Fisher, 1:200). Fluorescently-conjugated phalloidin was used to label F-actin (Alexa Fluor 488 or Alexa Fluor 568 phalloidin, Thermo Fisher).

All chemicals not specifically mentioned are from Sigma-Aldrich (highest grade). All inhibitors not specifically mentioned are from Cayman Chemical. The following antibodies were used: Alexa Fluor 647 anti-human CD3 antibody (UCHT1, BioLegend), Alexa Fluor 488 anti-human Granzyme A antibody (CB9, BioLegend), Alexa Fluor 647 or Brilliant Violet (BV) 510 anti-human perforin antibody (dG9, BioLegend), Alexa Fluor 647 anti-human granzyme B antibody (GB11, BioLegend), Brilliant Violet (BV) 421 anti-human CD178 (Fas-L), anti α-Tubulin mAb antibody (DM1A, Cell Signaling Technology), and Alexa Fluor 405 conjugated goat anti-mouse IgG (H+L) cross-absorbed secondary antibody (ThermoFisher Scientific). All isotype controls of fluorescence conjugated primary antibodies are from BioLegend. The following reagents were used: Hoechst 33342 (ThermoFisher Scientific), Alexa Fluor 488 or Alexa Fluor 568 phalloidin (ThermoFisher Scientific), Atto 488 NHS ester ((ThermoFisher Scientific), collagenase type I (ThermoFisher Scientific), FibriCol^® type I collagen Solution (Bovine, Advanced Biomatrix).