Cell centrifugation at 3200xg - Can cells survive such speed? (Apr/23/2007 )

I wander what happens to the cells when centrifugated at 3200xg?

This corresponds to 4000 rpm using the Eppendrof a-4-62 rotor, 18 cm radius.

Usually I spin at 1000 rpm (210xg), however there was a suggestion to use 3200xg in order not to loose the apoptotic cells (sub-G1 phase) which are lighter then alive cells and probably are lost from the pellet. Googling the web I also saw that some protocols do use such high speeds during washing.

Is there anybody here who has used such high rcf with success?

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Try it - spin some cells, mix 10 ul with trypan blue and count the blue colonies ... since live cells exclude the dye you can figure out what proportion survived.

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Dear maarts,

It is well known that centrifugation kills cells. I spin my cells after trypsinisation at 100g. What you propose is to spin them at 32 times that normal speed. You will capture your dead cells but you will also cause more cells to apoptose and die. Trypan is a viable/exclusion dye but will give you no information about cells going into apoptosis.

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Immediately after sedimenting the cells I am going to fixate them in 70% ethanol (for propidium-iodide staining). So, maybe that's why the guy suggested that high speed - because the cells should not stay alive anyway (?)

The problem is that I do not detect peak for chromatin at sub-G1 phase during flow cytometry (after overnight PI staining). Other cell cycle phases have nice peaks. So, most probably I am loosing the apoptitic cells somehow (I induce the apoptosis with cytokines), maybe during centrifugation.

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I usually spin cell media at 16000xg to pellet stray cells and dead/floaty stuff. I wonder, does a high centrifugal force squeeze out cell contents?

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