Re: Hoechst vs FITC: possible?
Howard Shapiro (hms@shapirolab.com)
Wed, 7 Feb 1996 19:56:51 -0500
Mike Loken did Hoechst/FITC immunofluorescence with collinear beams in a
paper published around 1980; he had to compensate the hell out of the FITC
signal (and didn't get all the Hoechst out even then).
>
> 1. There is published evidence of a long wavelength shift for
>unbound Hoechst relative to Hoechst bound to DNA. Should excess dye
>therefore be washed out of the final suspension? Note: these cells
>are fixed; is Hoechst retention the same for these as for viable
>cells?
>
> 2. How far can the Hoechst be diluted without prejudicing the
>stoichiometry?
>
In general, good stoichiometry can be maintained with 1 uM Hoechst; higher
concentrations have to be used with viable cells in order to counteract the
effect of the efflux pump. Dye not bound to DNA is at least 100x less
fluorescent.
> 3. Can the Hoechst emission spectrum be manipulated to shorter
>wavelength in any way (we have tried a longer FITC filter, 535DF15, but
>that didn't help much)? BTW, our Hoechst fluorescent beads from Flow
>Cytometry Standards give *much less* spillover!?
>
Do the beads have Hoechst bound to DNA or just a high concentration of Hoechst?
>
> 4. Are either of 33342 and 33258 superior in this context?
>
Probably not with fixed cells.
> 5. Is this experiment *obviously* impossible?
>
No, but it is at least difficult. Why do you need Hoechst in particular if
the cells are fixed, or are you constrained from using RNAse? And why is
not separating the beams so important?
--Howard
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