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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?

-canotto-

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-

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

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


Yes, I use the 10X binding buffer from BD biosciences. The Ca2+ is necessary for AnnexinV to bind.

-goldfinger-