Difference between revisions of "Primary Adipocyte Isolation"
From Bridges Lab Protocols
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# 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. | # 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. | ||
# 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. | # 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. | ||
| − | # Centrifuge at 500g ( RPM) for 10 minutes to separate floating adipocytes from pelleted SVF (pre-adipocytes and leukocytes). | + | # Centrifuge at 500g (2200 RPM) for 10 minutes to separate floating adipocytes from pelleted SVF (pre-adipocytes and leukocytes). |
# Gently aspirate the floating fat layer above the cells (if visible). | # Gently aspirate the floating fat layer above the cells (if visible). | ||
# 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.. | # 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.. | ||
# Aspirate remaining liquid, being careful not to disrupt the pellet. | # Aspirate remaining liquid, being careful not to disrupt the pellet. | ||
# Resuspend the pellet in SDS-lysis buffer or media (if performing experiments on the SVF) | # Resuspend the pellet in SDS-lysis buffer or media (if performing experiments on the SVF) | ||
| + | |||
| + | [[ Category: Adipocytes ]] | ||
| + | [[ Category: Metabolism ]] | ||
| + | [[ Category: Cell Culture ]] | ||
| + | [[ Category: Tissue Culture ]] | ||
Latest revision as of 14:57, 11 November 2014
The primary references for this is PMID
Materials
- 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.
Protocol
- Ensure that the microcentrifuge is at room temperature, not 4C.
- Check that the shaking water bath is set to 37C
- Excise fat pads from mice and mince thoroughly with small scissors in a weigh boat filled with KRBH, so that no chunks are visible.
- 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.
- 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.
- Centrifuge at 500g (2200 RPM) for 10 minutes to separate floating adipocytes from pelleted SVF (pre-adipocytes and leukocytes).
- Gently aspirate the floating fat layer above the cells (if visible).
- 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..
- Aspirate remaining liquid, being careful not to disrupt the pellet.
- Resuspend the pellet in SDS-lysis buffer or media (if performing experiments on the SVF)
