Isolation and ex vivo expansion of mouse FRCs.

Mice were sacrificed and blood was extracted by cardiac puncture. Omentum were collected. Mesenteric adipose tissue was carefully excised from the small intestine, large intestine, and cecum using scissors without rupture of intestines. Lymph nodes were removed. Pancreas was kept untouched. Omentum and mesentery adipose tissue were minced in RPMI 1640 Medium (Gibco) containing 2% FBS (Biotechne), 60 μg/mL Liberase TL (MilliporeSigma), and 250 μg/mL DNase I (MilliporeSigma). Minced adipose tissue was digested at 37°C for 25 minutes of shaking. Digestion was stopped by adding 4 times the volume of RPMI 1640 with 10% FBS. Digested tissue was filtered through a 70 μM sterile filter and then centrifuged for 5 minutes at 250g. Supernatant was discarded, and cells were resuspended in MesenCult Expansion medium (STEMCELL Technologies Inc.). Cells were cultured at 37°C with 5% CO₂ and used for in vitro experiments after 7 days.