annexin V and RBC lysing

rozenkov@netscape.net rozenkov at netscape.net
Sun Feb 19 01:39:55 EST 2006


Dear Indresh,
I have just come across a discussion on this Mailing List several years ago of direct
relevance to your recent question about RBC lysing and AnnV staining. A review was posted
on this list by Ruth Wilkins on 29 August 2001, so you can check it.
With your lysing solution, if you stain with annexin V first and then replace annexin
binding buffer with NH4Cl in water for lysing followed with washes, annexin can fall off
without Ca++ in the media.
We also use 7-AAD instead of PI. The positive staining is lower than with PI, but high
enough to distinguish. A good point about 7-AAD is that it is not such a nasty carcinogen
as PI. A possible advantage it that 7-AAD leaves the FL2/PE channel free (although some
people manage to use PI together with PE labelled antibodies).
A disadvantage for multi-color staining is that 7-AAD can occupy two channels (e.g., FL3
and FL4), and it is also a bit more expensive than PI (although may still be quite cheap,
especially if you do not buy it as a kit).
Regards.
Vladislav Rozenkov, MD, PhD
-----Original Message-----
From: IKaur at mdanderson.org
To: cyto-inbox
Sent: Tue, 31 Jan 2006 14:36:58 -0600
Subject: annexin V and RBC lysing



We are staining cells for AnnexinV and PI (using the kit from Pharmingen) to look for
apoptotic cells, but also need to lyse the RBCs. Should the RBCs be lysed before or after
the AnnexinV staining. We are using Pharmlyse ammonium chloride lysing solution.
Also, has anyone used 7AAD instead of PI. 


Indresh Kaur, Ph.D.
Coordinator, Flow Cytometry Lab
Department of Blood & Marrow Transplantation
713-563-4811 
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