On Fri, 07 May 1999, you wrote: >Hello everyone, > >Sraboni here. I'm new to the mailing list and a graduate student. I'd be >grateful for any experience/thoughts/ideas on the following: > >I'm using a FACScan to do a promoter readout assay with GFP, which I >transfect by electroporation in a megakaryocytic cell line. As an intrinsic >readout for rate of transfection my electroporation medium has 5. 10 -6 M >propidium iodide, the reasoning being that in a population gated for live >cells only those that reseal their membranes after the electroshock will be >PI positive. The problem is that in the condition where cells haven't been >tansfected but still kept in PI overnight, the live cell population has >about 1% PI positive cells. >The Molecular Probes catalogue says that PI is intact membrane impermeable, >so live cells should not be stained at all. If, as someone here says, these >cells are necrotic, shouldn't they become smaller than viable cells and >move to the left on the FCS axis? > >My question is: why are some live cells staining positive for PI? Could >anyone suggest another marker I could use as an intrinsic transfection >indicator which could be excited by an argon laser? > > I may be misunderstanding your question, but it seems to me that you are using FSC and SSC to determine whether your cells are live or not. (You say that 1% of "live cells" are PI-positive, and since you haven't said that you are using any other viability stain, I've assumed you are gating away the debris and aggregate tails from your FSC/SSC plot to find your "live" population.) PI is a much more accurate indicator of cell viability/membrane permeability than the FSC/SSC plot. I personally have seen many cases where a "dead" cell exhibits similar FSC and SSC characteristics as live cell, possibly for any one of a number of reasons. For one thing, FSC only correlates directly to particle size if the particle is completely spherical, which most cells are not. In summary, I would believe the PI-status of a cell over FSC readings when determining viability. Kelly Hardwicke Flow Cytometry Assistant Salk Institute for Biological Studies, La Jolla, CA
This archive was generated by hypermail 2b29 : Wed Apr 03 2002 - 11:53:28 EST