Davebrid: added categories and RPM

2014-11-11T14:57:58Z

added categories and RPM

← Older revision		Revision as of 14:57, 11 November 2014
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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 ]]

Davebrid: wrote initial page

2014-11-11T14:25:54Z

wrote initial page

New page

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 ( 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)

Primary Adipocyte Isolation - Revision history

Davebrid: added categories and RPM

Davebrid: wrote initial page