Primary Adipocyte Isolation

The primary references for this is PMID

• Collagenase, type I from Worthington
• KRBH Buffer, prewarmed to 37 in the water bath
• KRBH Buffer + 0.5% BSA and 1 mg/mL collagenase. Need about 1-2 mL per g of fat pad.

1. Ensure that the microcentrifuge is at room temperature, not 4C.
2. Check that the shaking water bath is set to 37C
3. Excise fat pads from mice and mince thoroughly with small scissors in a weigh boat filled with KRBH, so that no chunks are visible.
4. Add minced cells (~500 mg) to 1 mL of KRBH with BSA/collagenase in a 2 mL eppendorf tube. Wrap lids of tubes with parafilm to prevent leaking.
5. Incubate in shaking water bath for 20-30 minutes at 37C with vigorous shaking. Cells should completely disintegrate into individual cells, extend the time if necessary.
6. Centrifuge at 500g (2200 RPM) for 10 minutes to separate floating adipocytes from pelleted SVF (pre-adipocytes and leukocytes).
7. Gently aspirate the floating fat layer above the cells (if visible).
8. Using a cut 1000 uL pipet tip, very carefully remove the floating adipocyte layer to a fresh tube with warm KRBH (if performing further experiments) or SDS-lysis buffer..
9. Aspirate remaining liquid, being careful not to disrupt the pellet.
10. Resuspend the pellet in SDS-lysis buffer or media (if performing experiments on the SVF)