Primary Adipocyte Isolation: Difference between revisions
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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 ]] | |||