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
>