Ex vivo lipolysis

Ex vivo lipolysis of bone marrow adipose tissue

Reagents prep before dissection:
1. Culture media: pre-warm HBSS (Calcium and magnesium negative) and make 2% FFA free HBSS solution, filter it and ready for use.

2. Forsklin solution: Cayman (Item No. 11018), 50mg powder dissolved in 4.87ml DMSO, stock concentration as 25mM→ 1:10 dilution by DMSO (2.5mM)→ 1:500 dilution by culture media (5uM) for working solution.

3. Tubes for tissue transferring (on ice), tubes for flushing bones with media, tubes for culture media (with or without forsklin) and tubes for sample collection.

Steps:

1. Dissect mouse, cut distal tibia and small piece of gWAT as control, put in 2% BSA HBSS culture media on ice.
2. Transfer to cell culture room. Use 1ml syringe with 27G needle to flush the bone marrow in culture media.
3. Incubate in 37C with 5% CO₂.
4. Collect the supernatant at different time points for NEFA and glycerol measurements. 
5. Wash tissues by HBSS solution for 2-3 times→ lyse tissue and do protein assay to quantity the protein.