Protocols isolate cancer microparticles - Revision history

Michael C Larson at 02:42, 9 September 2011

2011-09-09T02:42:19Z

←Older revision		Revision as of 02:42, 9 September 2011
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	==Notes==		==Notes==
	Happy to have your positive input!		Happy to have your positive input!
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	'* The cells can be at a lower confluence and the incubation time can be longer. For example, I was able to get enough MPs for flow cytometry from 50% confluent cells after collecting the media that had been incubating the cells for 48 hours.		'* The cells can be at a lower confluence and the incubation time can be longer. For example, I was able to get enough MPs for flow cytometry from 50% confluent cells after collecting the media that had been incubating the cells for 48 hours.
		+
	'** Ultracentrifugation does something to the 'stickiness' of MPs. For example, centrifugation at 10000g at 10°C for 30 minutes is not able to create a visible pellet from MDA MB-231 cancer MP media obtained by this protocol. However centrifugation at 30000g at 10°C for 60 minutes of the same media created a visible pellet, after which resuspended, a 10000g spin at room temperature for 4 minutes resulted in a visible pellet.		'** Ultracentrifugation does something to the 'stickiness' of MPs. For example, centrifugation at 10000g at 10°C for 30 minutes is not able to create a visible pellet from MDA MB-231 cancer MP media obtained by this protocol. However centrifugation at 30000g at 10°C for 60 minutes of the same media created a visible pellet, after which resuspended, a 10000g spin at room temperature for 4 minutes resulted in a visible pellet.
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	[[Image:MDA_mps.jpg]]		[[Image:MDA_mps.jpg]]
-	<font face="courier"><nowiki>'''*~~MCL~~''':</nowiki></font>	+	<font face="courier"><nowiki>''*~~MCL~~'':</nowiki></font>

	==References==		==References==

Michael C Larson at 02:40, 9 September 2011

2011-09-09T02:40:08Z

←Older revision		Revision as of 02:40, 9 September 2011
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	'* The cells can be at a lower confluence and the incubation time can be longer. For example, I was able to get enough MPs for flow cytometry from 50% confluent cells after collecting the media that had been incubating the cells for 48 hours.		'* The cells can be at a lower confluence and the incubation time can be longer. For example, I was able to get enough MPs for flow cytometry from 50% confluent cells after collecting the media that had been incubating the cells for 48 hours.
	'** Ultracentrifugation does something to the 'stickiness' of MPs. For example, centrifugation at 10000g at 10°C for 30 minutes is not able to create a visible pellet from MDA MB-231 cancer MP media obtained by this protocol. However centrifugation at 30000g at 10°C for 60 minutes of the same media created a visible pellet, after which resuspended, a 10000g spin at room temperature for 4 minutes resulted in a visible pellet.		'** Ultracentrifugation does something to the 'stickiness' of MPs. For example, centrifugation at 10000g at 10°C for 30 minutes is not able to create a visible pellet from MDA MB-231 cancer MP media obtained by this protocol. However centrifugation at 30000g at 10°C for 60 minutes of the same media created a visible pellet, after which resuspended, a 10000g spin at room temperature for 4 minutes resulted in a visible pellet.
-	[[Image:~~MDA_mp~~.jpg]]	+	[[Image:MDA_mps.jpg]]
	<font face="courier"><nowiki>'''*~~MCL~~''':</nowiki></font>		<font face="courier"><nowiki>'''*~~MCL~~''':</nowiki></font>

Michael C Larson: New page: ==Overview== Microparticles or microvesicles are small blebs of membranes (typically defined as 0.1 to 1 or even 1.5 um in diameter, although this definition is arbitrary and under debate...

2011-09-09T02:38:02Z

New page: ==Overview== Microparticles or microvesicles are small blebs of membranes (typically defined as 0.1 to 1 or even 1.5 um in diameter, although this definition is arbitrary and under debate...

New page

==Overview==

Microparticles or microvesicles are small blebs of membranes (typically defined as 0.1 to 1 or even 1.5 um in diameter, although this definition is arbitrary and under debate) shed from numerous cancer cell types. The biological functions of these are under investigation 1.

==Materials==
#Typical cell culture lab equipment: sterile hood, 37°C incubator with 5% CO2, petri dishes, pipettes, centrifuge tubes etc.
#Cancer cell line--numerous types are available
#Media incubating cancer cell line supplemented with FBS (aka FCS; optimal % FBS varies by cell type) and optional antibiotics (i.e. Penicillin/Streptomycin)

==Equipment==
#Centrifuge capable of 1500g (optional cooling to 10°C)
#Ultracentrifuge if desiring to wash

==Procedure==
# Change media when cancer cells are approaching 75% confluence.
# Collect the incubation media after 24 hours* into a centrifuge tube.
# Centrifuge at 500-1500g for 10 minutes.
# Collect the from the top 3/4th to 4/5th of the media, discarding the pellet of loose cells/large debris. DO NOT simply dump from the top, unless you don't mind having to repeat step 3.
# Centrifuge again at 1500g for 15 minutes, collect from the top, discarding the pellet
## Ultracentrifuge at 20000g for 10 minutes to pull down large MPs. A longer/harder spin is required for pulling down smaller MPs, but for flow cytometry, this is enough to isolate MPs smaller than most flow cytometers can measure3.
## Wash and resuspend pellet in PBS or whatever else you want.**
# Snap freeze over liquid nitrogen and store in -80°C for long-term (weeks to months), -20°C for shorter term (days)

==Notes==
Happy to have your positive input!
'* The cells can be at a lower confluence and the incubation time can be longer. For example, I was able to get enough MPs for flow cytometry from 50% confluent cells after collecting the media that had been incubating the cells for 48 hours.
'** Ultracentrifugation does something to the 'stickiness' of MPs. For example, centrifugation at 10000g at 10°C for 30 minutes is not able to create a visible pellet from MDA MB-231 cancer MP media obtained by this protocol. However centrifugation at 30000g at 10°C for 60 minutes of the same media created a visible pellet, after which resuspended, a 10000g spin at room temperature for 4 minutes resulted in a visible pellet.
[[Image:MDA_mp.jpg]]
<nowiki>'''*~~MCL~~''':</nowiki>

==References==
1. Semin Thromb Hemost. 2010 Nov;36(8):888-906. Microparticles in cancer. Rak J. PMID 21049390

2. Platelets. 2008 Aug;19(5):365-72. Flow cytometric measurement of microparticles: pitfalls and protocol modifications. Shah MD, Bergeron AL, Dong JF, López JA. PMID 18791943

3. Transfus Med Rev. 2006 Jan;20(1):1-26. Cell membrane microparticles in blood and blood products: potentially pathogenic agents and diagnostic markers. Simak J, Gelderman MP. PMID 16373184

==Contact==
*Who has experience with this protocol?

or instead, [[Talk:{{PAGENAME}}|discuss this protocol]].

[[Category:Protocol]]

[[Category:Needs attention]]

[[Category:In vitro]]