Lynn Dustin wrote- >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? I hesitate to ask, but, since you noted in (4) above that you checked for debris, single cells, and aggregates by microscopy in the cells prepared for flow cytometry, did you notice whether the distribution of the two antigens was the same in these cells as in those prepared specifically for fluorescence microscopy? Clearly, the preparation of the cells for flow involves compromises, and I'd want to rule that out as a cause of loss of staining specificity before going any further. Next guess would be that the PE-conjugated secondary used for flow might have a harder time getting to the cytosolic antigen than would the Texas red-conjugated secondary used for microscopy. -Howard
This archive was generated by hypermail 2b29 : Sun Jan 05 2003 - 19:25:56 EST