Protocols isolate cancer microparticles

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

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

Equipment

1. Centrifuge capable of 1500g (optional cooling to 10°C)
2. Ultracentrifuge if desiring to wash

Procedure

1. Change media when cancer cells are approaching 75% confluence.
2. Collect the incubation media after 24 hours* into a centrifuge tube.
3. Centrifuge at 500-1500g for 10 minutes.
4. Collect the from the top 3/4^th to 4/5^th 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.
5. Centrifuge again at 1500g for 15 minutes, collect from the top, discarding the pellet
   1. 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.
   2. Wash and resuspend pellet in PBS or whatever else you want.**
6. 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.

''*~~MCL~~'':

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

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