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 ---------- From: owner-cyto-sendout[SMTP:owner-cyto-sendout@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 monograph 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 aqueous solution of 7-AAD since I read Ingrid Schmid's Cytometry article in 1992. In fact the last batch ( 50 ml made in PBS with azide @ 25 ug/ml and used at 1 ug/ml ) was made in 1995 -- stored at 4=BAC in the dark has no precipitate 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 me 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= solutions. Brent Dorsett chief, flow cytometry facility Lenox Hill Hospital, NYC =20
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