I am trying to find a protocol for measuring apoptosis in lymphocytes in whole blood (1:10 dilution with RPMI) samples using flow cytometry. I' looking method for lysing the red blood cells without damaging the lymphocytes. I am measuring apoptosis using Annexin V and am also looking specifically at CD4 and CD8 T-cells. I have tried using the recommended Ammonium Chloride procedure and I have also tried PharM Lyse from BD and find that both greatly reduce the viability (to about 75%) although the flow plots look good. However, I don't seem to get any Annexin V binding using the standard labelling concentration. I am also currently trying a water lysis method in which the cells are equilibrated with 1.8% NaCl 30 seconds after adding water. With this method the viability remains high (about 96%) but I still don't get any Annexin V binding in the lymphocyte population. The erythrocytes seem to be taking up all the Annexin V. Has anyone ever used the water method for lysis and if so, how were the results. I am hoping someone might have some experience with these methods and might be able to provide some advice. Thanks Ruth Wilkins, Ph.D. Consumer and Clinical Radiation Protection Bureau Health Canada 775 Brookfield Road, PL 6303B Ottawa, ON K1A 1C1 (613) 941-7263 FAX (613) 941-1734
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