Freezing spleen cells; with or without red blood cell lysis buffer?

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• 
  Warren Mikulka · Regeneron

  Hi- In my experience, freezing splenocytes before the usual red-cell lyse step is much better than lysing first and then freezing.The presence of the serum, etc in the splenocyte suspension before being washed away in a lysed step, helps to protect the cells in the freeze/thaw process. We have also found it is not essential to lyse after thawing back the splenocytes, In fact it is probably better not to, since the freeze/thaw process itself will naturally disrupt the delicate red cell membranes, and then the splenocytes can be washed after thawing. Warren Mikulka

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  Hi-
  In my experience, freezing splenocytes before the usual red-cell lyse step is much better than lysing first and then freezing.The presence of the serum, etc in the splenocyte suspension before being washed away in a lysed step, helps to protect the cells in the freeze/thaw process. We have also found it is not essential to lyse after thawing back the splenocytes, In fact it is probably better not to, since the freeze/thaw process itself will naturally disrupt the delicate red cell membranes, and then the splenocytes can be washed after thawing.
  Warren Mikulka

  Mar 13, 2012
• 
  Nuria Pascual ·

  Thank you very much!! Do you think that obtimum splenocytes concentration in freezing media could be 10 to the power of 7?

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  Thank you very much!! Do you think that obtimum splenocytes concentration in freezing media could be 10 to the power of 7?

  Mar 14, 2012
• 
  Warren Mikulka · Regeneron

  One entire mouse spleen splenocyte suspension pellet resuspended in 1 -1.5 ml freezing media , and then frozen in one 1.8 ml cryovial worked best for me throughout the years.

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  One entire mouse spleen splenocyte suspension pellet resuspended in 1 -1.5 ml freezing media , and then frozen in one 1.8 ml cryovial worked best for me throughout the years.

  Mar 20, 2012