930
edits
Changes
added categories and RPM
# 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)
[[ Category: Adipocytes ]]
[[ Category: Metabolism ]]
[[ Category: Cell Culture ]]
[[ Category: Tissue Culture ]]
