SMP isolation and culture in osteogenic media

Subcutaneous progenitor cell isolation

1. Remove the hairs from the mouse skin with electric hair remover and then put the mice in 70% ETOH for 10-15 mins.
2. Remove the skin from the body and keep it in cold sterile PBS on ice.
3. Subcutaneous skin tissue containing adipose deposits was removed under sterile conditions and washed for 4 times in PBS supplemented with 100 U/mL penicillin and 100 μg/mL streptomycin on ice (Figure1). 
4. The tissue was then cut to small pieces and minced, and digested with 1 mg/ml collagenase type I and 0.5%Trypsin in 0.1% BSA for 70 minutes at 37 °C. 
5. The digested tissue was centrifuged at 500 g for 10 min and the pellet was carefully collected after aspirating off the floating fat depots. 
6. After a second centrifugation at 500 g for 10 min, the cellular pellet was filtered through a 100-μm mesh filter to remove debris. The cells were seeded and cultured in αMEM with 20% FBS.

                                         Figure1