Mary-Lou, One hour should be more than enough for most cell types. Some cell types are a bit more resilient, but again, one hour is usually plenty. Of course, we're talking at least 50% ethanol. Re. apoptosis . . . it has been suggested that extending fixation to 24 hours improves resolution of sub-G1 cells. We have seen this to be true. Since cells can be kept in ethanol for extended times without consequence, we often tend to recommend 24 hours as a standard. If you're seeing variation in staining, I would look at your cell counts as a more probable source of that variation. Of course, using an internal standard (trout RBCs) will often eliminate this as a problem. MAK. >>> Mary-Lou Zuzarte <ZUZARTE@IMT.Uni-Marburg.DE> 10/09/01 17:27 PM >>> Dear flow users, I would like your opinion to a situation concerning fixation of cells for DNA analysis. We are working with a number of human cancer cell lines in the study of cell cycle and apoptosis. We have found that a 1 hour fixation with cold EtOH is not enough to obtain an adequate DNA profile. We have, to be sure that the protocol is consistantly followed, fixed the cells overnight. I think That 1 hour is not enough because we see massive variations in the instrument settings of the Calibur. The cells are stained with PI. I would really appreciate some feedback to this opinion. Thank you in advance, Marylou Zuzarte FACS & Confocal Facility Prof. Rolf Muller, Inst. f. Molekularbiologie & Tumorforschung Emil-Mannkopf Str 2 35037 Marburg, Germany Tel: 49-(0)6421-2865925 Zuzarte@IMT.uni-marburg.de
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