5 replies to this topic

[annacarrasco]

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

[miBunny]

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

[annacarrasco]

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

[miBunny]

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.

[illuminated]

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.

[vinayadass]

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