What exactly seems to be your problem? I am doing similar kinds of work with PI and have found on a FACSCalibur that I actually have to aquire the data twice, once with the channel for PI in Linear mode and once with it in Log mode. The linear settings just don't give enough of a separation for analysis against other flurochromes in Log mode. I am use to linear mode for DNA analysis so I need that to get results I understand. I hope this helps. Email me if the problems are otherwise. Janet Dow >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 Janet Dow Research Technician and Manager Flow Cytometry Facility North Carolina State College of Veterinary Medicine Room C-314 Raleigh, NC 27606 (919)513-6364
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