Cd49f bv421

Isolation methods were adapted from Strand et al.^{20 (link)} Briefly, samples were minced to a paste in the digestion medium (25 U/mL Collagenase type I (Gibco, Thermo Fisher), 0.25 mg/mL Dispase (Gibco) in RPMI 1640 medium with 10% FBS). Using 5 mL per 200 mg tissue, samples were incubated overnight in 50 mL tubes at 37°C with shaking and were spun down at 200 g for 5 minutes. Samples were next incubated in TrypLE Express (Life Technologies, Thermo Fisher) for 5 minutes at 37°C before being neutralized in RPMI 1640 medium containing 10% FBS, and passed through 18G needles to 70 μm cell strainers. Samples were incubated in ACK (ammonium-chloride-potassium) lysis buffer for 5 minutes at RT before flooding with phosphate buffered saline (PBS) and spinning down. Single-cell suspension was incubated in Zombie Viability Dye (BioLegend) for 10 to 15 minutes and spun down. Antibody cocktail (CD45-FITC, EpCAM-PE, CD26-APC, and CD49f-BV421, 1:100 dilution; BioLegend) was added to cells followed by incubation at 4°C for 30 minutes in the dark. Cells were filtered and sorted on BD FACSARIA (BD Biosciences, San Jose, CA) for luminal (CD45−, EpCAM+, CD26+, and CD49f−) and basal (CD45−, EpCAM+, CD26−, and CD49f+) cells. Sorted cells were washed with PBS and immediately suspended in TRIzol Reagent (Thermo Fisher) for subsequent RNA and protein isolation according to manufacturer's protocols.