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Double "mirror" lymphocyte population


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5 replies to this topic

#1 annacarrasco

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Posted 22 July 2009 - 04:49 AM

Hi everyone,

I have problems with my cell culture/activation/staining protocol. I analyze the samples (cultured PBMCs or freshly isolated mucosal intestinal lamina propia lymphocytes) with a FACSCalibur cytometer. and frequently I find a double lymphocyte population: one is smaller (200-400 FSC) and the other bigger (400-600FSC). they have the same expression pattern (regarding CD8, CD45 and CD3). usually the bigger population has few CD4 compared with the small one.

Is that normal? Am I doing something wrong with my cells?
Thanks

#2 miBunny

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Posted 23 July 2009 - 05:27 PM

it sounds like "doublets" (two cells attached to each other). We used to see these in mouse lymphocytes samples

#3 annacarrasco

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Posted 24 July 2009 - 01:41 AM

Thanks! How can I avoid or reduce that phenomenon? It normally increases with a long time culture.


it sounds like "doublets" (two cells attached to each other). We used to see these in mouse lymphocytes samples



#4 miBunny

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Posted 27 July 2009 - 05:52 PM

I honestly don't have a great method. I would try to pipet the cells to break them apart when you get them out of culture and gently vortexing them before running on the Facs.

#5 illuminated

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Posted 30 July 2009 - 05:10 AM

Thanks! How can I avoid or reduce that phenomenon? It normally increases with a long time culture.


Additionally to thoroughly mix the cells with the pipette, you could gate the doublets out for analysis.

#6 vinayadass

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Posted 25 August 2009 - 06:40 AM

5 mM EDTA/PBS/10 min at 37oC. Wash before using. This treatment will dissociate cell clumps generated in culture.




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