Nebe-Von-Caron, G g.nebe-von-caron at spdspark.com
Tue Nov 18 05:09:40 EST 2008

Considering that DAPI is used to stain viable bacteria




I would be careful about any interpretation of signals as Ethidium
Bromide was also used as a dead cell marker in mammalian cells and can
be loaded into bacteria but is subject to dye extrusion systems.
Normally cells with permeabilised membranes will always be quicker to
pick up staining so your brightest cells at low conc. or short
incubation times could be the dead ones but depending n circumstances
the dimmest fluorescence could be cells pumping the dye out or have
reduced amounts of nucleic acid. There is no way to get around
appropriate controls. Also a look of scatter versus fluorescence to get
some clues.



Gerhard Nebe-von-Caron 
Research Scientist and Biomedical Engineer 

SPDspark, www.swissprecisiondiagnostics.com

SPD Development Company Limited

Priory Business Park

Stannard Way

Bedford    MK44 3UP


Company Registration No.  6032177

mailto:g.nebe-von-caron at spdspark.com 



From: pearly.yong.j.a at nhc.com.sg [mailto:pearly.yong.j.a at nhc.com.sg] 
Sent: 16 November 2008 11:28
To: cyto-inbox
Cc: Cytometry Mailing List
Subject: Re: DAPI


Dear Rachael,


I've recently tried using DAPI as a a live/dead cell discriminator which
is excited off our UV laser (Aria). I've stained the cells for 15 mins
on ice (Final concentration of DAPI in cell suspension is 2.5ug/ml). I
am also interested in the questions which you have raised in your post
as we are relatively new to using DAPI for such purposes. 



Dear Flowers,


For the unstained sample, i observed two histogram peaks where the
second peak ends at the third decade log. For DAPI stained cells, i
observed that this whole peak shifts to the right (with the first peak
starting from the third decade log and the second peak showing up more
at the fifth decade log). Is this shift towards the right what others
observe when they stain for DAPI? Also on a DAPI vs SSC plot, i observed
three clusters of cells. 


Am i right to say that the ones to the furthest left are the viable
ones, while those in the middle are the apoptotic ones and those at the
far right end are the dead ones? Please correct me if i am wrong. All
advice is greatly appreciated!


Many thanks,



-----Rachael Walker <rvw24 at cam.ac.uk> wrote: -----

To: cyto-inbox
Date: 11/14/2008 10:01PM
Subject: DAPI

Dear all

I have been recommending to people in my lab to use ToPro -3 with their 
experiments on our CyAn and MoFlo for live dead discrimination and it 
has been working well.	  Last week I had a 561nm laser fitted to my
and I have to remove my 635nm red diode to use it, therefore people 
can't use ToPro 3 as the live dead marker.  As I work mostly with 
embryonic stem cells, a live dead marker is essential.	  As I
it PI will also be excited by the 561 nm laser (I haven't tried it as I 
am using my 613/20 PI filter to look at RFP while I wait for a new 
610/20 filter to arrive) and therefore is a no go.  I have some Sytox 
green but most people have GFP positive cells.	  Which has led me to
people to use DAPI as their live/dead discriminator using my 351nm 
laser.	  Twice today I have asked people to use DAPI and both times
have said  no because they thought DAPI was too harsh on cells and kills

them.  I assumed that DAPI worked the same as the other live dead 
markers such as PI, in that you have to have a breakdown of the membrane

for the dye to get in, but if you put far too much in then it will be 
toxic to cells.

I have looked DAPI up on the internet and found on Wikipedia (which I 
don't always believe hence the email) that DAPI is taken up by both live

and dead cells.  I have also done a quick search on the Purdue list and 
have found quite a few old emails about DAPI but they are referring more

to using it for cell cycle and the concerns about UV light on the 
viability of cells.

So my questions are, is DAPI a good live/dead marker?  Is it more harsh 
to cells than PI etc? What conc to people usually use?	  Is there a 
hierarchy of live/dead markers that people usually use?

Many thanks


Dr Rachael Walker
Flow Cytometry Core Facility Manager
Wellcome Trust Centre for Stem Cell Research
University of Cambridge
Tennis Court Road

01223 760227


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