Dear Flow-ers, I've one question about annexin V staining and its possible influence & effect on scatter characteristics of cells. I try to determine the amount of apoptotic cells by annexin V staining kit (PharMingen) and subsequent analysis on a FACScan. The conditions used in my experiments comprise incubation with apoptosis inducing antibody CH11 (Immunotech) and a variety of cytokines e.g. TNF-alpha. The cells I use for my investigations are human leukemic B-cells (mature B immunotype). On analysis of the cells, I experience a change in scatter characteristics (see ASCII depiction below). All prior experiments (e.g. CD95 expression, bcl-2 expression) showed a typical scatter picture of lymphocytes or lymphoblasts with a medium forward scatter and a low side scatter. Staining with annexin V causes the cells to have a low forward scatter and a medium side scatter so as the dot-plot had been mirrored. This phenomenon occurs on all conditions analysed (including untreated control). I checked for possible staining procedure errors, but I didn't find anything to have gone wrong according to the kit's description and procedure manual. Has anyone else on the list experienced such a change in scatter characteristics ? Could this scatter change be due to the binding buffer necessary for annexin staining (my suspicion, but unproven yet) ? Any help on this problem is greatly appreciated. Thanks in advance Best regards, Symbolic picture of normal scatter Symbolic picture of changed scatter S ! S ! I ! I ! D ! D ! E ! E ! * ! ! *** S ! S ! **** C ! C ! ***** A ! A ! ****** T ! *********** T ! ******* T ! ********** T ! ****** E !--------------------------> E !-----------------------------> R FORWARD SCATTER (FSC) R FORWARD SCATTER (FSC) If anyone needs more precise data on this problem, I can mail the data files. Thanks again _________________________________________________________ Andreas Christaras Heinrich-Heine University Children's Hospital Duesseldorf Lab. of Experimental Hematology (KMT-Labor) Bldg. 14.82 (Gebaeude 14.82) - Moorenstrasse 5 4 0 2 2 5 D U E S S E L D O R F - G E R M A N Y --------------------------------------------------------- Fon: +49-211-81-16103 Fax: +49-211-81-16191 E-Mail: christar@uni-duesseldorf.de _________________________________________________________
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