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We would like to measure intra-cellular FITC vs Hoechst-stained DNA
and we *have done this* with the exciting beams (488nm, UV) spaced
apart but, for reasons of the wider experiment, we hoped to do it now
with colinear beams. On our first attempt, we found the Hoechst
fluorescence swamping the FITC signal (far too much for fluorescence
compensation to accurately handle, even when exciting the Hoechst
with as little as 10mW UV). We would love to have advice from anyone
(particularly if you have tried this experiment, with or without
success). In particular:
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?
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!?
4. Are either of 33342 and 33258 superior in this context?
5. Is this experiment *obviously* impossible?
Thanks for *any* comment.
\ / < Flow Systems Laboratory
Frank Battye \__/ <<<<< The Walter & Eliza Hall Institute
Robyn Muir ----------!!<<<<<<<< Voice: 61_3_9345 2540
Dora Constantinou /!!\ <<<<< Fax: 61_3_9347 0852
o !! \ < IN:: "facs_copy@wehi.edu.au"
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