Intestinal stem cells and Paneth cell isolation was performed as described previously (Igarashi & Guarente, 2016 ; Roth et al., 2012 : Yilmaz et al., 2012 ). Briefly, the crypt suspensions were centrifuged for 5 min at 250 g at 4°C. The pellets were gently resuspended in 1.0 ml of undiluted TrypLE Express (Life Technologies) +120 μl of DNase I (10 U/μl, Roche). The suspended crypts were incubated in a 32°C water bath for 1.5 min were then placed on ice. 12 ml of cold MEM was added, and crypts were gently triturated twice. After centrifugation, the pellets were resuspended and incubated for 15 min on ice in 1 ml MEM containing CD45‐PE (1/500 eBioscience), CD31‐PE (1/500 Biolegend), Ter119‐PE (1/500 Biolegend), and CD24‐Pacific Blue (1/500 Biolegend). After centrifugation, the pellets were resuspended with MEM containing 1.5 μM propidium iodide (PI) (Thermo Scientific). The samples were filtered through a 40‐μm mesh (Corning) and immediately sorted on a FACS Aria (Becton Dickinson).
Intestinal stem cells were isolated as Lgr5‐EGFPhiCD24low/−CD31−Ter119−CD45−PI−,and Paneth cells were isolated as CD24hiSideScatterhiLgR5‐EGFP−CD31−Ter119−CD45−PI− (Igarashi & Guarente,2016 ).
Intestinal stem cells were isolated as Lgr5‐EGFPhiCD24low/−CD31−Ter119−CD45−PI−,and Paneth cells were isolated as CD24hiSideScatterhiLgR5‐EGFP−CD31−Ter119−CD45−PI− (Igarashi & Guarente,