Kathleen Schell wrote: > Does anyone know if a live-dead excluder like Propidium iodide or 7-AAD > could contaminate a sort of live cells enough to interfere with mRNA > measurements or PCR? Even after washing? We have an investigator who > insists on not using any dyes except monoclonal antibodies during sorts of > fresh tissue. We have had difficulty excluding dead and dying cells based > on scatter and autofluorescence and would realllllly like to include a dye > to help us. Any suggestions or comments? > Thanks. > Kathy Kathy,Truth be known, light scatter is perhaps the worst way to RELIABLY eliminate dead cells from your sort. If you do use a viability marker, you'll see that quite often there are significant numbers of events in the region that we normally call "live." The opposite is true as well, where changes in light scatter properties having nothing to do with viability can cause population to fall into "dead" regions erroneously. Therefore, if you want to eliminate dead cells from your mix, you either 1) make sure the viability is ~100% up front, or 2) use an exclusion dye. We routinely add PI (0.5-1ug/ml) to sort samples, and have had no problems with reactivity, clonogenicity, mRNA/protein extraction . . . whatever. MAK. -- Mark A. KuKuruga, Managing Director University of Michigan Core Flow Cytometry kukuruga@medmail.med.umich.edu
This archive was generated by hypermail 2b29 : Wed Apr 03 2002 - 11:53:22 EST