I want to decide which dye to use to detect actual mitosis (not just DNA syntesis or cycling) in a highly purified subset of T cells. Does anyone has experience using both CFSE and PKH26? What's best? Since the cells are purified, I don't need to do much additional phenotyping, ohter than determining DNA integrity.
I've never used PKH26, but I used an alternative to CFSE called "Oregon Green 488", which is much less sensitive to pH-mediated decreases in fluorescence, as its pKa is ~4.6. It seemed very stable, and the dye was detectable in the cells for several days (unsure how many generations). If you're doing FACS, I would include markers of apoptosis (i.e. Annexin V) and cell cycle or DNA labeling (Krishin's stain) if possible, as these could be helpful in quantifying proliferation-related changes.
Invitrogens Violet Tracker is also supposed to be less toxic than CFSE. I haven't tried this myself though.
I often use CFSE for my proliferation assays but in association with a viability marker like 7-AAD so that, during the acquisition, making a gate in order to exclude dead cells, I'm sure to see only proliferating live cells.
I am working with proliferation dyes and as far as I know CFSE & eflour670 are the best ones.
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