I've been doing some work with buffy coats lately and using ACK lysing solution. I sometimes have to repeat the procedure to get rid of all RBC, but it doesn't seem to be affecting either surface or intracellular staining. ACK Solution To 1 litre of distilled water add: 8.29 g NH4Cl (Ammonium Chloride) [.15M] 1.0 g KHCO3 (Potassium Bicarbonate) [1 mM] 0.367 g Na2-EDTA [0.1 mM] Adjust pH to 7.2 to 7.4 with 1 N HCl. Filter through a 0.22m filter and store at room temperature or 4°C. Pellet cells in a 15 cc tube and decant supernatant. Resuspend pellet in at least 5 volumes of cold ACK Solution, and incubate at room temperature for 5 minutes. Top up tube PBS or HBSS to wash and spin as usual. Repeat if necessary. Paula Lavery Transplant Immunology Laboratory Royal Victoria Hospital McGill University "If we knew what we were doing it wouldn't be called research, would it?" -- Albert Einstein Al Sabirsh wrote: Hello to all, Does anybody have any suggestions regarding lysis of red blood cells contaminating buffy coat preparations? I'm inclined to use distilled water because I want to use the polymorphonuclear cells in a subsequent chemotaxis assay (so anything with formaldehyde in it is out of the question). I've tried a number of different approaches, but the buffy coat preparation is very thick and seems to be resistant to hypotonic shock (even after dilution in 0.85% saline or PBS, or prolonged incubation at low salt concentration). Separating out rbc's using density gradients (Ficoll-Iso/Hypaque, before or after attempted lysis) doesn't work at all, but I was thinking of using dextran to get them to clump together or something (sort of like anti red cell mAbs). I also have a couple of protocols using weak Tris buffers, vinager or ammonium, but I thought I'd ask the list because I want something that won't affect the function of the polymorphonuclear cells. So does anyone have experience with this? Should I just give up and use whole blood? (I wouldn't get as many cells that way though....) Thanks for the help, Al Sabirsh One of these grad students who know nothing about whats going on their test tubes. Now where did I put that cell lysis kit?... (sorry, couldn't resist that :-)
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