Has anyone had any suggestions for Anja Mindermann's gap junction problem? I have worked with a few people doing cell-cell conjugation experiments; a popular approach was to load one cell type with calcein AM and load the other cell with PKH26. On the other hand, the target cell could be surface stained with a PE-Ab, as long as the incubation between the two cell types isn't long enough for capping or shedding to be a significant factor. Neither of these approaches do exactly what Anja is trying to accomplish, in terms of detecting the exchange of calcein across a gap junction, resulting in the creation of calcein-only stained cells. Perhaps one could stain the donors with both calcein and a PE-Ab for a intensely expressed surface marker. Again, it would be important to minimize loss of the PE-Ab, which could appear as false positive "single stained" cells. If the scatter patterns of the two cell types were significantly different, that could be useful, too. I don't recall how PKH26 is different from or similar to DiA/DiI, so if they didn't work PKH26 may or may not be useful. What an interesting problem! -- Mark A. Miller University of Pennsylvania School of Dental Medicine Flow Cytometry Facility
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