The problem with membrane associated dyes like PKH26 or labeled cell surface antigens is the potential of cell-to-cell exchange that can occur--especially if gap junctions are present. One thing that might be tried is to use EMA (ethidium monoazide bromide, available from Molecular Probes). As I discussed before, we use it as a live/dead discriminant. However, even live cells will take up the EMA and fluoresce; if you use more than 5 ug/ml, then the live cells will be come quite bright. The EMA is then photocrosslinked to DNA by exposure to a regular desk lamp, and thus should be permanently associated with the DNA. The only problem is that the cells might not divide successfully (on the other hand, they might!). If you're not doing a long-term incubation then even this caveat would not be important. The final advantage to EMA is that you could still distinguish live cells from dead cells (dead at the beginning of the experiment, that is). EMA has a fluorescence spectrum similar to PI and can be excited on the 488; thus the FACSAnalyzer could detect it with only an emission filter change. Brief protocol (modify as you see fit): incubate cells 10 min with 5 ug/ml EMA (or more) in the dark. Wash a couple of times; expose in short proximity to a light source (like a desk lamp) for 10 min. Wash a couple more times, then use the cells! mr
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