Isolation and Characterization of Adipose-Derived Stromal Cells

ADSCs’ isolation was performed as previously reported [21 (link), 22 (link)]. Mice were killed by cervical dislocation and asepticized by soaking in 75% ethanol for 5 min. The inguinal adipose tissues were withdrawn and washed with PBS at 4 °C for three times. After removing the blood vessels and lymph nodes carefully, the adipose was cut into pieces and digested for 90 min at 37 °C using 10% fetal bovine serum (FBS) and 125 U/mL collagenase (Cat# 17018029, Collagenase Type I, Gibco) [23 (link)]. By filtrating through 100-μm nylon filter mesh (Cat# 352360, BD Falcon) and concentrated by centrifugation at 300g for 5 min, the stromal vascular fraction (SVF) was attained [24 (link)]. Then, ADSCs were resuspended in complete medium (high-glucose Dulbecco’s modified Eagle’s medium (H-DMEM) with 10% FBS and 1% penicillin-streptomycin solution (Cat# C0222, Beyotime, China)) and cultured in a humid atmosphere with 5% CO₂ at 37 °C. The culture medium was updated every 48 h. The cells were monitored daily under an inverted phase-contrast microscope (Leica DMI4000 B) and passaged when they reached 80–90% confluence.
Cells at passage three were used for 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) (Cat# C1157, Invitrogen) labeling and identification according to the standard listed in Additional file 1.