Al, when I was doing proliferation and NBT assays, I would dilute my whole blood with 1 ml 6% dextran/ 10 mL blood, let settle for about 45 minutes in 37C CO2 incubator, and then ficoll the plasma layer. If I wanted the mononuclear cells, they were very clean. For PMN's I would resuspend the pellet in Hank's and add 3 times that volume of distilled water, shake for 60 seconds, followed by an al volmue equal to the total volume of 3.5% NaCl. The cells worked beautifully. Good luck. Candace Enockson, enockson@musc.edu --On Sat, Mar 20, 1999 6:30 PM +0100 Al Sabirsh <Alan.Sabirsh@mphy.lu.se> 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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