From: /G=Gerhard/S=Nebe-von-Caron/OU=1890CHPE/O=TMGB.URC/@LANGATE.gb.sprint.com
Date: Tue Aug 01 1995 - 06:01:00 EST
Looking at dye uptake in bacteria I found similar discrepancies for
the uptake of ethidium bromide (EB). Apart from pumps removing the dye
that can be overcome with sodium azide in most cases, penetration is
sometimes very slow. The simplest trick to enhance uptake is to stain
in a very high concentration initially (>100ug/ml) and dilute
subsequently. Make sure you keep the final DMSO concentration down
(stocks in PBS) as that can improve uptake but also cell damage. The
addition of 0.05% tween 20 even at low EB concentrations (<5ug) allows
staining within 15 minutes, whilst propidium Iodide (PI) does not.
We use a combination of EB and PI together with FITC labelled antibody
to sort bacteria. We have not yet obtained exact data on the tox
effects of the DNA stain, but the longer they incubate in EB, the less
bacteria grow on the plates.
Gerhard Nebe-v.Caron
Unilever Research
Colworth Laboratory
Sharnbrook, Beds.,
UK MK441LQ
email: gerhard.nebe-von-caron@urcgb.sprint.com
______________________________ Reply Separator _________________________________
Subject: DAPI
Author: Alice.L.Givan@dartmouth.edu at INTERNET
Date: 29/07/95 19:48
Lots of good replies on DAPI! Thanks.
Consensus (if you can call it consensus) seems to be that DAPI may or may not
get into living cells -- depending on the type of cell (membrane
characteristics/ glycoprotein pumps) and the patience of the investigator (that
is, DAPI penetrates slowly at best). In some cell types and with some
protocols , it doesn't penetrate living cells very well and therefore it can
be used to differentiate living and dead cells. In cases where it does get
into living cells, it will stain DNA -- but will probably not give as pretty
DNA histograms as Hoechst 33342 (although some people report better success on
this than others). Hoechst 33342 seems to be, by acclamation, the dye of
choice for looking at ploidy in living cells. Questions were raised about DAPI
not leaving the cells viable after the staining even if it does penetrate the
membrane; this is obviously a question that needs to be considered (along with
mutagenesis) for any dye that enters a living cell and binds to its DNA....
For fixed or permeabilized cells, consensus is that DAPI and PI both give good
DNA histograms.
Thanks for all your help.
Alice Givan
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