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
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