Live dead fixable zombie yellow fixable viability kit

Tumors were minced, chopped, and digested with Gentle Collagenase, Dispase 0.012% (w/v) and DNaseI (STEMCELL Technologies) at 37 for 1 hour. Single cell suspensions were obtained by passage through a strainer (70 µm), washed in FACS buffer (PBS with 5% FBS), incubated with LIVE/DEAD Fixable Zombie Yellow Fixable Viability Kit (Biolegend, 423104) for 30 min., and then blocked with anti-CD16/32 (Biolegend, clone 93) for 5 min. on ice. Samples were then incubated with primary fluorophore-conjugated antibodies on ice for 45 min. For detection of intracellular markers, FOXP3 Fixation/Permeabilization Buffer Set (BioLegend) was used, according to the manufacturer's instructions. Antibodies for flow cytometry are described in the Star Methods. Flow cytometry was performed on an LSR II flow cytometer at the Flow Cytometry Core of the PCC Precision Immunology Shared Resource and analyzed with FlowJo software. Organoids cultured 6 days after infection with MSCV-Kras-mCherry were collected and digested as above, passed through a strainer (70 µm) to obtain single-cell suspensions, centrifuged at 1000×g for 5 min, and resuspended in PBS containing 2% FBS, 10 µM Y-27632, (STEMCELL Technologies Inc.), and DAPI (1 µg/ml). FACS was performed immediately on a MoFloTM XDP, and mCherry hi and mCherry neg cells were seeded at 5,000/well.