RPM for Pelleting down cells - (Jul/16/2008 )

I need to spin down the media with cells so that I need only the media (supernatant) without any live cell in that. I spin the media with cells at 10,000 RPM for 10 min. It is extremely important for me that NOT even a single cell comes in the media. After spinning, I pipette soup carefully and avoid last ~50-100ul of media from the tube. Still is there any chance that I might take some cells in to the media which might still be alive and proliferate after this 10K spin for 10'?? (I do not want to waste 0.2u filter everytime)

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10,000 may be harmful for cells; to guarantee absence of cells in the supernatant, it will be better to filter (0.2 µm pore size)

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The first big question is, Calvin*, do you care about the cells you're pelleting? If you don't, then you can spin as fast as you want. If you do, you'll want to keep the RCF low. I've heard 2000 xg is the highest you want to go with eukaryotic cells, but I don't have a citation to give you on that.

My personal experience with pelleting is that the slightest bump will cause some of the pellet to disperse. Normally this is negligible, but since you're going for no cells at all in the media, this may not be sufficient for you. Higher RCFs may decrease this, but I doubt that any benchtop centrifuge will be able to eliminate this effect. As The Bearer said, you're probably better off using .2 um filters. As far as cost-cutting measures go, these may or may not be reusable - I don't do much filtration of large volumes, so you'll have to ask around on that.

Also, NO ONE can tell you the proper RPM to use, since we don't know what what rotor you are using. Instead, all we can give you is the Relative Centrifugal Force (RCF), measured in multiples of g, which you should then be able to convert to the RPM needed for your rotor.

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Nice call Squire! You are absolutely right that RPM means nothing without rotor information. It is much better to get used to talking in terms of RCF which can be converted for any rotor.

If your entire experiment hinges on the fact that you have no remaining cells in the media, it is most definitely worth the cost of a disposable 0.2um filter. Much cheaper, I'm sure, than the media and reagents and time it will take to repeat the experiment if even one cell remains!

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