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I'm working with murine B-cells. The general protocol is to keep cells on ice to keep them from dying but I've noticed that it makes these cells aggregate and precipitate out. I've heard suggestions that these cells should just be kept at room temperature. How would I be able to determine which conditions at which I should be keeping my cells?

I was trying to collect cells to prepare them for cell binding studies to antibodies and then assess the cells on a filterplate. Typically, you keep them on ice to pause the metabolic state as well as to prevent endocytosis of the bound molecules. However, that shift seems to cause settling. How do you troubleshoot the appropriate temperature to process cells and what are the usual scientific justification for making such decisions.

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I know I'm not supposed to ask for clarification here, but I think it's completely necessary in order to provide an appropriate answer. Could you please clarify what you're doing with your cells? i.e. do you need to keep them physiologically active? How long are they out of the incubator? If cells are just going to FACS, ice is fine, just pulse the pellet prior to reading, a little EDTA can also help keeping them from clumping. In general, I would recommend ice or 37 degrees most of the time. –  Dr. T Jul 30 '13 at 21:56
    
@Rhill45, really? would say that part of the answer does involve "how long can you keep cell on ice". For instance, if you're going to FACS, what processing steps can you do before running them through the machine, what is the rate of aggregration, etc –  bobthejoe Feb 6 at 22:49
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Also, why are people collapsing questions from 2 years ago? –  bobthejoe Feb 6 at 22:51

1 Answer 1

For the settling there's little you can do except agitation and using a taller thinner tube and larger suspending volume.

For the clumping however if the analysis permits it, adding serum to a [final] of 5% will help slow the clumping (in the short term). You can also filter out initial clumps by pushing the cells through a nylon mesh filter (they make these especially for flow-commonly used with fixed cells). I've analyzed cells on ice in dark probably as long as 6 hours after harvesting with little change in viability, however this is of course bad practice.

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