Cd69 bv711

Spleens from the indicated mice were collected and dissociated, followed by filtering through a cell strainer in PBS with 1% FBS. Cells were collected by centrifugation at 1500 rpm for 5 min, red blood cells were removed by red blood cell lysis buffer, and the remain cells were resuspended and incubated with antibodies (CD3-BV421, CD4-PECy7, CD8-BB515, CD69-BV711, Ly6c-BV786, CD44-APC, CD62L-PE, all from BD Biosciences) on ice in the dark (or wrap in foil) for 20 min. The strained cells were resuspended in PBS and analyzed with FACSymphony™ A5 instrument (BD Biosciences).

The indicated target cells were stained with Cell Trace Yellow following the manufacturer’s recommended protocol (ThermoFisher). Cells were then stained with the indicated antibody adaptors/amounts and were washed twice with flow cytometry buffer and resuspended in cell culture media. 10,000 target cells per well were then co-cultured with 50,000 mSA2 CAR T cells (effector:target=5:1) in a 96 well V-bottom plate. All co-incubation experiments with mSA2 CAR T cells were performed in DMEM media supplemented with 10% FBS. For UV-cleaving experiments, wells were then exposed to 365 nm light using a 365 nm LED (Mouser, 416-LST101G01UV01). For small molecule cleaving experiments, the indicated amounts of 2DPBM were added to wells containing all cell populations. Cells were then incubated at 37°C for 24 hours. Cells were then evaluated stained with fluorescently labeled antibodies recognizing T cell activation markers: CD69-BV711 (BD Biosciences) and CD107a-APC (BD Biosciences) and GFP for NFAT-induction experiments. Marker expression was specifically evaluated on mSA2 CAR+ by gating for the TagBFP+ population. Supernatants from primary cell assays were also collected and analyzed for IFNɣ by ELISA (BioLegend).