> 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!?
There is some evidence that suggests that the actual DNA/Hoechst complex
itself is very much cell type dependent, and is intimately related to the
DNA-phosphate ratio and the pH. The reference I'd point you at is Smith et
al. 1985 Flow- cytometric detection of changes in the fluorescence emission
spectrum of a vital DNA-specific dye in human tumour cells. Exptl.Cell.
res.159;37-46
With this in mind it may be worth playing with the pH, such that it is
optimized for FITC (since it's also pH dependent) and perhaps modifying the
phosphate concentration , say in the PBS in which the cells are suspended,
such that a Hoechst signal dampening occurs. I don't know if this is
possible, but it may be worth a try. I too think the spectral shift for
H333XX is concentration dependent, but as to the lowest "working" limit, I
haven't a clue.
I also recommend reading the section in James Watson's "Introduction to Flow
Cytometry" (Applications in Oncology : Therapy selection) , which could be
helpful.
Hope this helps, and if not, I've only recycled a few electrons "wastefully"!
Geoff
======================================================================
Geoffrey Osborne | ____ __ o Ahh!
Flow Cytometry (FACS LAB) | __ `\ <,_
John Curtin School of Medical Research, | __ (*)/ (*)
Australian National University, | ==============|
CANBERRA, AUSTRALIA. | |--|
Email: Geoff.Osborne@anu.edu.au | |--|...
Phone: 61 6 249 3694
FAX: 61 6 249 2595
-----Surfing the Web?: Try http://jcsmr.anu.edu.au/facshome.html------
======================================================================