> ---------- > From: Ruth_Wilkins@hc-sc.gc.ca > Sent: Thursday, 9 August 2001 3:22 > To: Cytometry Mailing List > Subject: lysing red blood cells before Annexin V labelling > > Ruth, > I have used FACSLyse from BD on whole blood cultures (1:2 with RPMI) and > stained with Annexin V without compromising lymphocyte viability (Hodge et > al. Increased levels of apoptosis of leucocyte subsets in cultured PBMCs > compared to whole blood as shown by Annexin V staining: relevance to > cytokine production. Cytokine 2000, 12, 1763-1768). Are you keeping plenty > of Ca++ in the staining mixture. I havent used the water lysis method. > > Greg Hodge PhD > Haematology Department, > Women's and Children's Hospital > 72 King William Rd > North Adelaide, > South Australia 5006 > ph: 08 82046522 > fax: 08 82046043 > email: hodgeg@mail.wch.sa.gov.au > --------------------------------------------------------------- > > > 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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