Two suggestions: 1. Instead of growing the cells in PI, resuspend in PI just before FACS analysis. You also might check the optimal concentration of PI (1 micro-molar should be enough). But 1% PI+ cells from a culture is not at all unusual. I completely agree with the comments below that FSC is not an accurate indicator of viablity. 2. If your GFP signal is bright enough, it will overlap into your PI channel (I assume you are using FL1 for GFP and FL3 for PI). If you gate away PI+ cells, you may gate away GFP+ cells. You can check by looking at gated and ungated data. The solution is either software compensation or using PI excitation (or another dye such as Hoescht 33342) by a laser that does not excite the GFP, if you have access to a dual laser instrument. Kelly Hardwicke wrote: > 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
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