One additional warning: we have found that CFSE at slightly high concentrations (5 uM) inhibits antigen specific T-cell proliferation. The PKH dyes do not appear to have this effect -- perhaps because they are labeling lipids rather than proteins. ConA-induced proliferation did not appear to be as sensitive to CFSE as did tetanus-induced proliferation. As Ryan Hung says, the problem with using low concentrations of CFSE is that you cannot let your cells divide for as many generations before they reach background fluorescence levels. Therefore, you cannot make use of the amplification in sensitivity that occurs as a small population of cells replicates through 6-8 generations. That sensitivity was important to us as we calculated precursor frequencies for small numbers of proliferating antigen-specific T-cells (J. Imm. Methods: 230: 99-112 (1999) with erratum, J.Imm. Methods 237: 207). So, getting the right concentration of either CFSE or PKH26 is tricky: too high and you get compensation problems and also potential functional inhibition; too low and you lose sensiivity. 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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