Hi everybody,

I have to centrifuge my cells (fibroblasts) several times for a FACS staining and my problem is that I lose most of them during the procedure. I fix them in 70% ethanol, then centrifuge at 2000rpm/5min. Ater this I still have a pellet, but at the later washing steps (same speed) it´s more and more vanishing. Does anybody have an idea what I could change? Longer centrifugation? another speed? other tubes? ( I am using normal falcons).

Thanks
dinah

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what are you washing with? solutions with greater density will require either longer or faster spins to have the same effect.

you should probably just spin longer. faster may damage your cells.

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I am washing with PBS and PBS with BSA and Tween (0.2%).

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these are more dense than 70% ethanol. in order to have complete pelleting of your cells you should increase the time that you spin. you can move up in increments until you find the least amount of time that you require to pellet all of your cells, then increase the time by 10%.

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we use 2500 rpm/10 to 20 min

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Thanks for your ideas and explanations, I centrifuge now for 15minutes and that works good enough.

dinah

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