Double "mirror" lymphocyte population - (Jul/22/2009 )

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

-annacarrasco-

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

-miBunny-

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

miBunny on Jul 23 2009, 06:27 PM said:

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

-annacarrasco-

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.

-miBunny-

annacarrasco on Jul 24 2009, 02:41 AM said:

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.

-illuminated-

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

-vinayadass-