Hi Lyndal Try the gentle ethanol treatment protocol described originally in Garvy et al. (Immunology 79, 270-277, 1993) and later reviewed in Telford et al. (J. Immunol. Meth. 172, 1-16, 1994). This method involves resuspension of the cells in 50% FBS in PBS prior to the gradual addition of a 3-fold amount of cold 70% EtOH. Following overnight incubation the cells can be washed from the FBS/ethanol and resuspended in PBS (with or without a DNA dye) for analysis. This method permeablizes the membrane and nucleus sufficiently to allow good DNA staining while causing minimal disruption of surface markers. We have used it extensively to simultaneously detect cell death in mouse lymphocytes using PI or 7-AAD simultaneously with FITC or FITC/PE immunophenotyping for surface markers. No paraformaldehyde fixation is necessary. Hope this helps! Bill On Fri, 2 May 1997, Lyndal Russell wrote: > > Hi everyone!! > > We are trying to quantitate surface marker changes simultaneously with DNA > cell cycle analysis (using PI). We have tried this using ethanol fixation, > but have lost the surface markers. > > Can anyone suggest a recipe for a better fixation method? > > Thanks all! > > Lyndal Russell > >
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