RBC Lysis and Apoptosis - does it interfere? (Mar/24/2010 )

Hi,

I want to analyze apoptosis on peripheral lymphocyte populations. I was wondering if it is better to do an Annexin V staining to splenocytes, PBMCs or to lymhpnode cells.
I have read that mechanical shrinking of tissues may induce exposure of phosphatidilserine on the other side of the membrane, so giving false positives ( and this would be the case of lymphnodes and partly of spleen). Ficoll gradient centrifugation (in the case of blood and eventually spleen) I think may result in loss of dying apoptotic cells, that will be floating over the lymphomonocytes ring.
Also, I don't know if red blood cell lysis may influence the frequence of apoptotic lymphocytes ( and this would be the case of spleen and peripheral blood), and/or if it is better to avoid interference in apoptosis to lyse RBC with ACK lysing buffer or with ddH2O.

Do you have any experience or suggestions regarding this assay?

I'd like also to ask somebody if you have ever used this binding buffer for Annexin V, as it is suggested in the datasheet of my AnnV/APC :"PBS with Ca2+ = add 0.33 g/l to PBS" instead of other buffers containing HEPES

canotto on Mar 24 2010, 08:04 PM said: