Double "mirror" lymphocyte population - (Jul/22/2009 )
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?
it sounds like "doublets" (two cells attached to each other). We used to see these in mouse lymphocytes samples
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:
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.
annacarrasco on Jul 24 2009, 02:41 AM said:
Additionally to thoroughly mix the cells with the pipette, you could gate the doublets out for analysis.
5 mM EDTA/PBS/10 min at 37oC. Wash before using. This treatment will dissociate cell clumps generated in culture.