I ran into a bizarre problem. Here is a representative figure. Each column indicates a cell line with or without lentiviral transduction (mentioned on the figure). The problem is, I see a separate and distinct population for DAPI-ve cells at the edge (marked by arrow) for cells lentivirally transduced only. For the non-transduced cells (basically the parent cell line), live cells (DAP-) showed up at its usual spot. These data were collected at the same time. Any help will be greatly appreciated
Without having worked directly with lentivirus, is it possible that it is altering the binding sites that DAPI would use. You seem to have more signal variation, alongside the negatives, in the lentiviral cell population
My guess would be dead/dying cells in the process of shearing their chromatin, hence the lower DAPI signal. You'll also notice that your FSC/SSC shows more FSC-lo SSC-hi/lo events, which is also indicative of increased cell death in culture. The DAPI staining pattern also changes very noticeably with your lentivirus-transduced cells as Lewis Williams pointed out. My suggestion would be to add a viability dye in this experiment to determine how drastically lentiviral transduction affects your cells. Also, did you treat the non-transduced cells with whatever transduction reagent you're using? In my experience, some cells/cell lines don't tolerate things like polybrene, etc. very well, so it might be worth looking at other reagents. Hope this helps.
-Max
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