>Date: Wed, 23 Apr 97 11:21:26 PDT >From: Tom Mc Closkey <thomasm@nshs.edu> >Subject: RE: Annexin V staining >To: Cytometry Mailing List <cytometry@flowcyt.cyto.purdue.edu> > > > >>I have recently been attempting Annexin V staining and so far this >>has been succesful. However I am worried about the amount of debris in the > >>bottom left of FSC/SSC, should this be included in the final analysis >>or excluded? >> >I guess the answer depends on whether the events w/ low FS ansd SS are >apoptotic cells or debris or some other cell type [depending on the system]. > For th e most part, the info you want is % apoptotic cells of a particular >population. So you need some method to determine whole cells [apoptotic or >live] from other events. If you can't achieve this with LS then maybe >adding a McAb or staining for DNA wil l be a required gating step. > >Good luck, >Tom Hi James, To add to Tom's answer, the events with low SSC and low FSC may depend on whether you have FSC and SSC amps set to log. If this is so then what you are really seeing is debris form your sheath fluid. You could try a linear scale for the FSC if this is the case. Tom suggests that you use an antibody to detect your cell population, if the scattering is cellular debris then your Ab may stick to it, giving you a false positive. A DNA stain is more useful. If you are doing apoptosis work then you may want to discriminate the necrotic cells from the apoptotic cells. Most people use PI to do just that. Andy Riddell PNAC Division MRC Laboratory of Molecular Biology, Hills Road Cambridge CB2 2QH U.K. tel: (01223) 402 218 fax: (01223) 412 178 e-mail: ar3@mrc-lmb.cam.ac.uk Andy Riddell PNAC Division MRC Laboratory of Molecular Biology, Hills Road Cambridge CB2 2QH U.K. tel: (01223) 402 218 fax: (01223) 412 178 e-mail: ar3@mrc-lmb.cam.ac.uk
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