Hi. We've been doing some staining for BrdU incorporation in GFP-expressing cells, and have been getting some strange results. I'm hoping someone out there might have a solution, or at least an explanation. The assay goes like this: - Plate cells (neuronal precursors) in a 24-well plate and infect with retroviruses carrying Gene X-IRES-GFP (with or without mitogens) - At 48 hours, pulse with BrdU - The next morning, harvest the cells (they're adherent, but a brief papain treatment gets them off) - Fix with 1-4% paraformaldehyde (2% seems to work the best, but we've tried them all) - Permeabilize with 0.2% Triton X-100 (or Tween-20) - Treat with DNase to expose BrdU epitopes (can't use HCl, because it kills GFP) - Stain with anti-BrdU (PE-conjugated) and anti-GFP (FITC-conjugated) - Analyze by FACS for BrdU and GFP expression Regardless of whether the virus is empty or contains something that should induce cell cycle progression, we get the same results: some cells that are GFP-positive/BrdU-negative, some cells that are GFP-negative/BrdU-positive, but none that are double-positive. The fact that we're getting both types of cells in a given sample suggests that the BrdU staining is working, and that the GFP hasn't leaked out of the cell. Another possibility is that the viruses (or GFP) are toxic to the cells, and any cell that has GFP is unable to proliferate. However, when I do essentially the same experiment using immunofluorescent (IF) staining of cells on coverslips, I get plenty of double-labelled cells. (The results of that assay are very clear, but much more difficult to quantitate, which is why we've been trying to do it by FACS.) The only differences I can come up with between the assays are: (1) for IF, the cells are growing on coverslips instead of on plastic; (2) for IF, cells don't get papained off before staining; (3) for IF, we use biotin-anti-BrdU + streptavidin-TRITC (rhodamine) instead of a PE-coupled antibody. None of these seem likely explanations for the dichotomy. Has anyone had this experience? Anyone want to take a stab at an explanation? Thanks very much for your help, Rob -- Robert Wechsler-Reya, Ph.D. Department of Pharmacology and Cancer Biology Box 3813, Duke University Medical Center LSRC Building, Room C303 Durham, NC 27710 Phone: 919-613-8754 Fax: 919-681-8461
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