Hoechst 33342 and FITC antibodies
Howard Shapiro (HPANDA@HARVARDA.HARVARD.EDU)
Thu, 05 Jan 95 12:46:44 EST
Davin Jutila asks whether UV excitation of FITC might contribute to the
Hoechst 33342 signal in cells stained with the Hoechst dye and FITC-
labeled antibodies. This is extremely unlikely if even approximately
the right filters are used for the Hoechst dye, since FITC shows negli-
gible emission in the range below 480 nm. The biggest problem in
staining live mouse lymphocytes with Hoechst dyes is the active efflux
of dye catalyzed by the glycoprotein drug efflux pump, which is highly
active in many mouse strains. Krishan showed that Hoechst dye efflux
could be blocked in lymphocytes from at least some strains of mice by
trifluoperazine or verapamil; Crissman et al found that staining with
cyanine dyes improved CV's of Hoechst dye fluorescence in live cells,
presumably due to the cyanines' inhibition of energy metabolism. Unless
you need to do live sorting, the easiest way to get good Hoechst dye
staining of mouse lymphocytes is to fix them. For live cells, you need
a high dye concentration, maintained through all wash steps, inhibitors,
and a substantial amount of luck.
--Howard
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