Flow Cytometry Mail Group FLOWUSER at cyto.medsch.ucla.edu
Wed Feb 5 14:55:00 EST 1997

Dear Brent,
I make up 7-AAD using 1mg by first adding 50 microliters of absolute   
methanol then adding 950 microliters of 1 x PBS.  Originally, when I   
started using 7-AAD for DNA staining in the late 1980s I was really   
worried about stability and change of staining properties of 7-AAD. Since   
then however,I have made the same observation as you did, that the   
solution when kept at 4 degrees covered with foil protected from light it   
can be used at least for dead cell discrimination and in our hands also   
for DNA staining for much longer than one month. This has been also the   
practice of the many users of our core facility who use 7-AAD for dead   
cell discrimination.

Ingrid Schmid

 owner-cyto-sendout[SMTP:owner-cyto-sendout at flowcyt.cyto.purdue.edu]
Sent:  Friday, January 31, 1997 3:30 PM
To:  Cytometry Mailing List
Subject:  7-AAD

Please excuse me for being a little behind the flow but I've had the
midwinter sniffles. I was just catching up on the recent live cell
discrimination dialog. In one of the replies was a reference to a   
from the University of Washington about using 7-AAD. Included in it were
instructions for making a solution in DMSO, since it is not water soluble
and storing 1 month at 4 degrees C because of limited stability. This I
confirmed with a call to Calbiochem. All this came as something of a
surprise to me since I have been using ( perfectly successfully ) an   
solution of 7-AAD since  I read Ingrid Schmid's Cytometry article in   
In fact the last batch ( 50 ml made in PBS with azide @ 25 ug/ml and used   
1 ug/ml ) was made in 1995 -- stored at 4=BAC in the dark has no   
and works just fine. Whenever some project calls for dead cell
discrimination in three color flow -- out comes the 7 AAD. Now don't get   
wrong -- I'm not suggesting that I'm right and others are wrong. I'm just
reporting the facts I've observed. Since that batch was made there have
never been any observable shifts in fluorescence intensity or required
compensation. Maybe ignorance has just been bliss. At any rate I would be
interested in whether anyone else has used 7AAD from  aqueous stock=

Brent Dorsett
chief, flow cytometry facility
Lenox Hill Hospital, NYC =20

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