Your leukapheresis sample is collected using ACD as the anticoagulant and will contain ~300mg% citrate ion. If you are experiencing coagulation, more than likely your buffers/reagents contain divalent ions, specifically Ca++ and Mg++, thus causing the coagulation due to the binding to the citrate ion. Change to reagents without Ca++ and Mg++ and your problem should disappear. April April G. Durett, MSc Clinical Applications - Flow Cytometry Center for Cell & Gene Therapy Houston, TX -----Original Message----- From: Guo Yalin [mailto:GUO@mm11.ukl.uni-freiburg.de] Sent: Thursday, September 28, 2000 6:04 AM To: cyto-inbox Subject: coagulation in leukapheresis Dear flows, My project is sorting SP cells, a kind of stem cells from human cord blood, bone marrow and leukapheresis with Hoechst 33342. During fresh leukapheresis cells were stained in Hoechst at 37 C, coagulation of cells always happened. It's not good for both of staining and sorting. I have tried with 1 mM EDTA and/or 1 U/ml DNase and didn't get any good result. Does anybody has such experience and has some solution to prevent the coagulation, which is definitely what I need? I should appreciate if somebody could tell me in detail soon! Thank you very much for any suggestion! Yours sincerely, Yalin Guo Yalin Guo Nothnagel Lab Hematology/Onkology Department University of Freiburg Hugstetter Strasse 55 D-79106 Freiburg Germany Tel: +49 761 270 7199 Fax +49 761 270 7177 Email: guo@mm11.ukl.uni-freiburg.de
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