In response to Michelle Miller's question (see below)... I think there is a very good chance that your transfected cell samples contain a lot of sub-cellular debris, especially if they have been through harsh chemical or mechanical manipulations. As useful as scatter is in flow cytometry, it is not going to be adequate to find a consensus with trypan blue exclusion. You might want to try Molecular Probes' Live/Dead viability kit, or make your own with PI and an fluoroscein-like esterase substrate stain, such as calcein-AM or Carboxy-2',7'-dichlorofluorescein diacetate (carboxy-DCFDA). A MUCH less rigorous approach would be to turn your forward scatter discriminator up a little. Get this... just yesterday I was emailing cytometry advice to my brother, Michael Miller! Mark Miller University of Pennsylvania School of Dental Medicine Flow Cytometry Facility > Dear Flowers, > > When doing transfection experiments, we routinely determine viability of > the samples by FACS on the basis of FCS vs SSC profiles (these are > non-adherent cell lines). Someone in the lab has just done an > experiment where they checked the viability of each sample by trypan > blue exclusion by light microscopy as well as by FACS. To her surprise > viabilities were much higher by trypan blue than by FACS (eg 60% vs 17%, > respectively). > > One way to explain this is that maybe non-viable cells break up so one > single non-viable cells becomes several "events" in the non-viable area > of a FACS plot, leading to a disproportionate number of non-viable vs > viable events. Does anyone else have any suggestions, or seen this > disceprancy themselves? Which is more accurate? I would always have > said FACS but now I'm not so sure. > > Thanks in advance, > Michelle >
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