I would think you are seeing autofluorescence contribution in your flow results. I am assuming your control samples on the flow were all fine and reasonable voltages were applied to the PMTs. Most microscopes use a dichroic and longpass filter configuration which will let autofluorescence through. Since our eyes have a difficult time distinguishing between intensities of diffuse versus punctate (we tend to overestimate punctate and underestimate diffuse) I would also suggest doing some image analysis with some digital images and looking at fluorescence intensities of cells. The only other thing I can suggest would be to look at the linear signal on flow for any sign of subtle changes otherwise lost in the Log signal. At 10:38 AM 1/15/02 -0500, you wrote: >Hello out there in flow land, > >I am trying to help a colleague who is examining expression of a cell >surface (endogenous) marker and a cytosolic (viral protein) marker. She >got different results by flow from those obtained by microscopy. >Expression levels of the viral protein vary from cell to cell in these >clonal populations (for interesting reasons which I am not at liberty to >discuss). By microscopy, she sees two distinct cell populations; the >expression of the cell surface and cytosolic antigens are mutually >exclusive. By flow, we expected to see the same 2 cell populations. >Instead, there was a single staining pattern and most cells appeared to >be stained for both antigens. > >We have confirmed that this is not a compensation issue. There does not >appear to be a problem of background staining or a cross-reactive >secondary antibody. We do not get this result with cells (different >clones, derived from the same parental line) that do not express the >viral protein. > >We are considering various technical reasons for this discrepancy. If >any of you have experience in this area, your >comments and suggestions are most appreciated. > >Possible technical sources of trouble? >1. The cells are adherent. For microscopy, she grew them on coverslips. >For flow, she grew them to the same density on plastic, and removed them >by two different methods: scraping or trypsin. Either method gave us the >same problem. She prefers to scrape the cells to avoid loss of the >surface marker. > >2. Both of the antigens are stained indirectly (unlabeled primary, >fluorochrome-conjugated secondary). The antibody combination for the >surface antigen is unchanged. For the cytosolic antigen, a mouse primary >is detected with Texas Red-conjgated 2° for microcopy, but a >PE-conjugated 2° for flow. We do not have much background staining, as >demonstrated by staining a matched clone that does not express the >target protein. > >3. Fixation and permeabilization: for microscopy, the cells were grown >on coverslips, fixed with 3.7% formaldehyde, and stained in the presence >of 0.1% Triton X-100. For flow, the scraped or trypsinized cells were >fixed with 2% paraformaldehyde, then permeabilized, stained, and washed >in the presence of 0.1% saponin. In each case, the protocols were >chosen because they have worked for that particular application in other >experiments. > >4. Gating: Cells were filtered before running on the cytometer. We were >able to detect cell debris, single cells and small aggregates (as judged >by forward and side scatter, and checked on the microscope). We gated >separately on the single cells and on the aggregates, with the same >results. > >5. Antigen distribution: When examined by microscopy, the cytosolic >viral antigen is localized in discrete regions of the cell. The punctate >distribution may make it easier to see small amounts of antigen. The >membrane antigen, by contrast, is distributed all over the cell surface. > >The cytometer is a FACSCalibur set up to use 4 colors (we routinely use >Alexa 488/fluorescein, phycoerythrin, PerCP, and Allophycocyanin at the >same time). The microscope is a Nikon Eclipse TE300 with filters for >fluorescein or Alexa 488, Texas Red, and Hoechst. > >Sorry for the long-winded letter. Any ideas on how to sort this out? >Many, many thanks in advance. >********************************************** >Lynn B. Dustin, Ph.D. >Center for the Study of Hepatitis C >Rockefeller University >Box 64 >1230 York Ave. >New York, New York 10021 >Phone: 212-327-7067 >FAX: 212-327-7048 >email: dustinl@mail.rockefeller.edu Derek Schulze Flow Cytometry and Confocal Microscopy Facility Manager Cancer Research Labs Queen's University Kingston, ON Canada http://meds.queensu.ca/medicine/crl/flow/
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