Ghost dye red viability dye

Cell coincubations were prepared
as described above for cell activation experiments except that following
24 h incubation, cells were stained with Ghost Dye Red Viability Dye
(Tonbo Biosciences) to calculate dead cells, and coincubations were
analyzed by flow cytometry. Target cells were identified by Cell Trace
Yellow or Cell Tracker Deep Red, and lysed target cells were identified
by positive Ghost Dye staining. Percent-specific cytotoxicity of target
cells was calculated by the equation: 100 × (% experimental lysis
– % target-only lysis)/(100 – % target-only lysis).

The indicated target cells were stained with CellTrace Yellow following the manufacturer’s recommended protocol (ThermoFisher), and 10,000 target cells per well were co-cultured with 50,000 SNAP-CAR T cells (E:T = 5:1) in a 96 well V-bottom plate with 1.0 μg/mL of the indicated BG-conjugated antibody. Plates underwent a quick-spin to collect cells at the bottom of the wells and were then incubated at 37 °C for 24 h. To identify lysed cells, co-incubated cells were stained with Ghost Dye Red Viability Dye (Tonbo Biosciences) and analyzed by flow cytometry. Target cells were identified by CellTrace Yellow and lysed target cells were identified by positive Ghost Dye staining. For the mixed target cell assay, Raji (CD20 + ) cells were stained with CellTrace Yellow. A total of 20,000 Raji, K562 + EGFRt, or Raji+K562-EGFRt (20,000 of each target) were co-incubated with 50,000 SNAP-CAR T cells and the indicated antibodies. K562 + EGFRt cells were identified by their membrane mCherry expression (EGFRt-[GGGGS]x3-mCherry). Percent specific cytotoxicity of target cells was calculated by the equation: 100*(% experimental lysis – % target-only lysis) / (100 – % target-only lysis). A representative plot summarizing the CAR T cell and target cell co-incubation gating scheme is shown in Supplementary Fig. S19.