Glass bottom culture 35mm plates

SEE (Toxin Technology) loaded Raji B cells (1.5×10⁶) were mixed with Jurkat T cells (1.5×10⁶), plated on poly-L-lysine (5 μg/ml) coated glass bottom culture 35mm plates (ibidi), and subjected to brief centrifugation prior to imaging. Images were taken with Zeiss 700 confocal microscope and quantified for conjugate formation as previously described (10 (link), 22 ).

Murine splenocytes were cultured with 1 ug/mL SEE and 1000 U/mL mIL-2 (Miltenyi) for 72 hours, followed by isolation of CD8 T cells using isolation kit (Miltenyi). SEE loaded Raji B cells (target cells) were mixed with CD8 T cells at different ratios, as indicated, and cytotoxicity was tested using LDH cytotoxicity kit (ThermoFisher). In some experiments, CD8 T cells were stained with Lysotracker (ThermoFisher) before mixing with Raji cells, plated on poly-L-lysine (5 μg/ml) coated glass bottom culture 35mm plates (ibidi), and subjected to brief centrifugation prior to imaging. Images were taken with Zeiss 700 confocal microscope and analyzed with ImageJ (NIH).