RE: DRAQ5 staining of bacterial cells *Industry input*

Dear Dongjuan

It would help me to know what concentrations you have tested DRAQ5 at. 
I suspect you meant 1ul/ml to 20 ul/ml rather than ug/ml since it is
supplied as solution of molar concentration.  
Also, can you tell me what bacterial cell concentration you have been
using?

For DRAQ5 the concentration of dye and cell (ie DNA load) are key
parameters, reflecting the fact that a dynamic equilibrium is being
established albeit massively in favour of the "on" component.  For
mammalian cells DNA content is 6pg DNA/cell compared to ca. 1/600 of
this in bacteria.  For mammalian cells we calculate on 5-20 uM DRAQ5
with a max. cell load of 4x10e5/ml.  I would translate that to a maximum
bacterial concentration of 2.5x10e8/ml (or 2.5x10e5/ul).  In mammalia,
staining is best at 20-37 degC though this might be lower for bacteria
depending upon their host environment.	 

Further to Gerhard's useful comments, DRAQ5 is optimally excited at 646
nm though the HeNe line is close to the absorbance maximum too.  In
mammalian cell studies DRAQ5 can be successfully excited with 488
Argon-Ion laser as described in numerous papers and concomitantly with
FITC, PE (& ECD ) on a single laser benchtop

However, I should add that the labeling of DRAQ5 as "toxic" relates to
our responsible attitude to labeling the hazard to the user; we follow
the benchmark guidelines of ANSI. This is not a standard adhered to by
all... ethidium bromide, etc would also fall into the category of
"toxic". 

For the cells themselves, like ethidium bromide and all DNA
intercalating dyes, DRAQ5 will obviously have an impact on replication.
There is good data from a recent Cytometry paper (R. Martin, Cytometry
Part A 67A:45-52 (2005)) that describes the impact on HeLa cell
viability 24 hours after staining with 1uM DRAQ5.  Cells remained viable
though they accumulated in G2/M.  This paper also explores the excellent
correlation between H2B expression and DRAQ5 cf. Hoechst 33342.

Gerhard is right in his observation about drug resistance / pumps.
DRAQ5 is generally not pumped out making it a timing-independent stain
for live cells.  A poster presented at SETG 2003, Barcelona by
Garcia-Escarp et al describes the efflux of Hoechst 33342 but not DRAQ5
in studies on drug-inhibition of ABCG2 transporters in human cells.

I hope this helps.
Please respond to the questions at the top of this mail so I can assist
you further.

Very best regards
Roy Edward
Biostatus Ltd.
-----Original Message-----
From: Nebe-Von-Caron, G [mailto:g.nebe-von-caron@unipath.com] 
Sent: 19 December 2006 17:30
To: cyto-inbox
Subject: [microbiology] RE: DRAQ5 staining of bacterial cells

Hi Dongjuan

Having obtained a small sample of DRAQ5 from Roy Bongaerts at the IFR
last week I have just played a bit with the dye. As Howard said, signal
is poor, but does exist. On the Elite I excite with the HENE and measure
with a 675nm band pass. I might improve things with a different filter
(long pass to catch more signal) but my current configuration I get a
much better signal to noise using ethidium bromide with the 488nm laser
and a og630 (schott) longpass. Only for the EB staining I have to
deenergize or block the pumps which does not seem to be necessary with
the DRAQ5. Exciting DRAQ5 with the 488 does not yield enough signal.

According to Advanced Analytical DRAQ5 gives a better s/n than Syto 62.
They use a 639nm laser diode but I do not know their filter combination.
However I can not confirm that by lack of Syto 62 to play with.

When it comes to sorting DRAQ5 stained cells you might have a problem as
the dye is said to be toxic, but again, I have not had time to
investigate that.

Good luck 

Gerhard




> -----Original Message-----
> From: Dongjuan DAI [mailto:daidj00@gmail.com] 
> Sent: 14 December 2006 15:34
> To: Cytometry Mailing List
> Subject: DRAQ5 staining of bacterial cells
> 
> 
> Hi Everyone,
> 
> Have you ever used this dye, DRAQ5 from Biostatus, on 
> bacteria? I tried it on both E.coli K12 and Pseudomonas 
> aeruginosa. But I cannot get any bacterial cells stained. Do 
> you have any ideas why I fail? I tried from 1ug/ml to 20ug/ml 
> of DRAQ5.
> 
> Thank you,
> Dongjuan
> 
> -- 
> Dongjuan Dai, Doctoral Student
> Dept. of Environmental Health Sciences
> School of Public Health
> the University of Michigan, Ann Arbor
> 109 Observatory St. 6645 SPH Tower, MI 48109
>