Difference between revisions of "Lipolysis Assay from Primary Adipocytes"

Latest revision as of 14:51, 11 November 2014

Materials

• Primary Adipocytes (see Primary Adipocyte Isolation )
• KRBH prewarmed to 37C
• Isoproterenol (stock made up at 10 uM, final use at 100 nM)

Protocol

• Set heating block to 37C
• If preparing adipocytes by collagenase digestion, gently wash cells 2-3x with warm KRBH to remove residual collagenase
• Incubate cells 1h in KRBH to equillibrate
• Prepare control or isoproterenol (diluted to 120 nM) assay mixtures by combining the drug with KRBH. You will need 500 uL for each assay. Aliquot this volume into the assay tubes
• Add approximately 100 uL of adipocyte/KRBH suspension (50:50 dilution in KRBH) to the assay tubes.
• Incubate for desired time (normally ~1h at 37C on the heating block) with occasional mixing (every 10 mins or so).
• Separately, using a hemocytometer, calculate the number of cells in the 100 uL aliquots (to normalize for potential differences in cell size).
• At the completion of the assay remove the liquid layer underneath the cells (~200 uL) to a fresh tube and place on ice. Be careful not to get any cells.
• If desired, aspirate remaining liquid and resuspend the cells in SDS media to perform western blots on the post-treated adipocytes.
• Assay the glycerol or free fatty acids using our standard protocols (see FFA and Glycerol Determination). Use KRBH as the blank.
• If not assaying the glycerol/FFA levels right away heat the samples at 70C for 10 minutes to inactivate lipase activity.
• Present the data in glycerol (or FFA) released per hour per number of cells.