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"We have a scientist who uses DAPI to discriminate between live and dead cells
(as with PI). However, Molecular Probes say that DAPI is a cell permeant DNA
stain (like the Hoechst dyes) and will stain living cells. Is this just a
kinetics difference so that DAPI enters living cells just a bit slower than it
enters dead cells? Has anyone used DAPI as an alternative to the Hoechst dyes
to stain living cells for ploidy? We want to be able to sort living cells on
the basis of their DNA content -- for further culture. Any suggestions?
Thanks!
Alice Givan
Englert Cell Analysis Laboratory
Dartmouth Medical School
Lebanon, NH 03756, USA
tel: 603-650-7907
fax: 603-650-6130"
The use of nuclear stains to assay cell viability is based on exclusion.
However, this exclusion should not be taken too literally. All these
nuclear dyes will eventually penetrate cells and bind to DNA. High dye
concetrations and long incubation times will speed up this process.
For example, PI is said not to penetrate viable cells, but it does somehow
induce petite mutants in yeast cells, so it must get into these cells.
We have used DAPI, PI, EtBr and Ho58 as exclusion dyes in mammalian and
microbial systems and obtained very similar results. Your scientist is
doing the correct thing as long as the cells are kept on ice and are not
incubated with DAPI for too long. In case of long sorts it pays to take
small aliquots of the sample to be sorted and add DAPI prior to hooking
them up.
In an extensive effort to generate decent DNA-profiles from viable yeast
cells only Ho42 penetrated sufficiently. Ho58, PI, EtBr and several other
gave some increased fluorescence but no DNA related signal.
So in my view you are stuck with Ho42.
But beware, this dye may 1) be pumped out of your cells (use verapamil) and
2) may be reduce the viability of the sorted cells.
Good luck,
Jan
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