Centrifugation after staining with PI and other DNA fluorochromes is not necessary for several reasons. 1) PI bound to DNA has a quantum efficiency at least 50 times that of PI free in solution. 2) The concentration of PI in stained nuclei is much higher than that in solution. 3) The flow cytometer is effectively looking at a volume of a few thousand femtoliters or less, so it won't pick up that much background fluorescence. 4) The electronics in the PMT preamplifier remove the DC background contribution from the fluorescence signal. Reason 1 applies to PI, ethidium, the chromomycin dyes, 7-aminoactinomycin D, DAPI, the Hoechst dyes, styryl dyes such as LDS751, and the monomeric and dimeric cyanines of the TO-PRO, TOTO, and SYTO series. Other dyes, such as oxazines and acridine orange, do not increase quantum efficiency markedly on binding to nucleic acid and may actually be quenched somewhat; background fluorescence with these is higher but reasons 2-4 still work. Reasons 2-4 also explain why one can do immunofluorescence without washing using directly labeled monoclonal antibodies. -Howard
This archive was generated by hypermail 2b29 : Wed Apr 03 2002 - 11:49:55 EST