Hello Vincent, Let me first take the time to say that one year ago today I was the one asking the same question. I can happily now give what I can say has been the easiest, most reliable manner I have found in doing whole blood. : 1- NH4CL: seems to work OK, although I've never checked pH. HOWEVER, it does not seem to be reliable...sometimes a pellet is left, and it also takes more time. 2- FACSLyse did not work well for us...also appeared somewhat unreliable in that sometimes a pellet would be left. There didn't appear to be a pattern, but perhaps there was something we were not doing (if so, I don't know what it was as we were following directions) 3- IOtest Whole blood Lyse and FIX (coulter/Immunitech) and Gentrak's similar product.....Gentrak's product is what we now use, but the two procedures are identical, although I believe that Gentrak's instructions are more detailed. As listed in the directions, you must vortex immediately after adding the lysing solution, therefore do NOT do more that 3 tubes at a time. Another thing we found that gave us a 100% lysing success rate is to vortex also JUST BEFORE adding the lysing agent. The lysing is done just after staining in these protocols. Patricia Echeagaray +++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++ P. Echeagaray Flow Cytometry Services Southern Research Frederick Frederick MD echeagaray@sri.org Vincent Gin wrote: > > To all of you who uses ammonium chloride lysing on peripheral blood and > bone marrow samples for surface markers: > 1. does the pH of the solution stay for more than 1 day? Ours goes from > pH 7.2 to as high as pH 7.7 on the next day. We have been making fresh > batch everyday. > 2. do you stain first, then lyse or lyse first? Are there any > differences in fluorescence intensity with one method or the other? > If there is anything else we should be aware of in using nh4cl lysing > vs FACSLyse, ficoll etc. please advise. Thanks much in advance! Nancy > Gin - United Hospital - St.Paul, MN.
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