I am having problem with a procedure for CD3/CD4/CD8 and DNA cell cycle analysis and wanted to see if anyone could help. Here is what I am doing (briefly) using mouse blood. 10 ul of CD3-Biotin + 100 ul blood incubate wash and spin 10ul SAV-ECD + CD8-FITC + CD4-FITC incubate wash and spin lyse with coulter whole blood lyse kit 60 seconds add 250 ul of the "fixative" in the whole blood lysing kit wash and spin wash and spin analyze for cell surface ( this is just a monitoring step) spin add 300 ul ot PBS + 700 ul of COLD EtOH 100% slowly dropwise incubate 15 minutes COLD spin resuspend in PBS - hydration 15 minutes RT spin add working solution of RNase (5 ug/ml) in TRIS(10 mM) and CaCL2 (5 mM) and 50% Glycerol buffer - 1ml add 20 ul of 7-AAD incubate 15 minutes Analyze I can use any help and if I am doing this all wrong, I am open to completely new procedures. Thanks in advance Bill Justice Laboratory Information Systems KU Medical Center
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