We have used both PKH26 and CFSE. Both are, with our cells, toxic at high concentrations and both seem less or not toxic at low concentrations. The trick, if you want to track many divisions, is that you want to start out with cells that are as bright as possible. So the titration window between getting bright cells and not killing them is critical. The scary thing we have found, particularly with CFSE, is that at somewhat high concentrations you inhibit some reactions (like antigen specific proliferation) but not other reactions (like ConA stimulated proliferation). So you do need to test with tritiated thymidine uptake under your own cell culture conditions to make sure that cell division is not affected by the CFSE or PKH labeling. >From first principles, it seems to me that it might be safer to use PKH dyes rather than CFSE because the CFSE will label proteins and may affect the function of some of them. Since the PKH dyes only involve lipids, the chance of specific toxicity might not be as great. But I know that many people have great success with CFSE --- and it certainly is much cheaper than the PKH dyes. Alice Alice L. Givan Englert Cell Analysis Laboratory of the Norris Cotton Cancer Center Dartmouth Medical School Lebanon, New Hampshire NH 03756 tel 603-650-7661 fax 603-650-6130 givan@dartmouth.edu
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