[Cytometry] False positive Annexin V?

Patricia Lovelace patricia.lovelace at sbcglobal.net
Tue Feb 9 20:57:04 EST 2010


Michelle,
What does the scatter look like on these cells? I ask because I have had 2 instances in which I was asked to troubleshoot why the scatter and annexin staining looked strange after staining with a kit and the problem was that the investigators did not notice that the annexin V binding buffer was supplied at 10x... so they used it at 10x, wrecking the cells. When they used it at 1x, everything was fine.
Patty Lovelacelovelace at stanford.edu
Manager, Flow CytometryStanford Stem Cell Institute1050 Arastradero Rd Palo Alto, CA 94304orHuman Immune Monitoring CoreCCSR 0128269 Campus Drive Rm 0128Stanford, CA 94305

--- On Mon, 2/8/10, Alireza <ardjmar72 at yahoo.com> wrote:

From: Alireza <ardjmar72 at yahoo.com>
Subject: Re: [Cytometry] False positive Annexin V?
To: "Mok Michelle" <michelle_mok at bsf.a-star.edu.sg>, "cytometry at lists.purdue.edu" <cytometry at lists.purdue.edu>
Date: Monday, February 8, 2010, 2:43 PM


Michelle!

Based on one of Shapiro's laws, all reagent vials don't necessarily contain what they should; so please check your Annexin with some other healthy cells eg. Lymphocytes and if possible induce them apoptosis by some thing like Dexametazone to see if your Annexin is working properly. I also think it worth to to checked the  DMSO effect on them as well; may be DMSO is just compromising the membrane integrity and not triggering apopotosis as its application in Cryopreservation though..
 

by the way I am not familiar with the HUH7 line but it should have some G0/G1 peak any way; doesn't it. and  you say there isn't any!!



 Kind Regard 
 
Alireza Ardjmand, M.Sc
PhD student

 
School of Biomedical Sciences & Pharmacy

Cancer Research Unit
University of Newcastle 

Callaghan, NSW 2308
Australia

 
Tel    +61 2- 49217954
Mob  +61  - 413286947
Fax    +61 2- 4921 6903
 






________________________________
 From: Mok Michelle <michelle_mok at bsf.a-star.edu.sg>
To: "cytometry at lists.purdue.edu" <cytometry at lists.purdue.edu>
Sent: Mon, 8 February, 2010 8:55:51 PM
Subject: [Cytometry] False positive Annexin V?

Dear all,

I have a puzzling situation. I have user who is trying to verify his apoptosis studies on HUH7 cells. Basically he has an untreated baseline, followed by 2 drugs in various concentrations, the idea being to see the different concentration of the drugs on cell apoptosis. He has done PI cell cycle analysis  via Flow Cytometry and Caspase studies by western blotting, the results from these 2 assays are correlating very well. However, when Annexin V assay is perform, the outcome is very strange.

The untreated baseline - DMSO, is showing no caspase cleavage in Western blotting and shows no G0/G1 peak during PI cell cycle analysis (meaning cells are all alive and OK, looks healthy on culture too), however, when Annexin V is perform, it is a strong positive. We believe this is probably a false positive, but we have no idea how to remedy it.


    |      |  Best Regards,
      \__/  Michelle Mok
  ------!!    Senior Application Specialist | Flow Cytometry | Biopolis Shared Facilities
      /!!\    TEL +65 6407 0148 (Office) | +65 6407 0147 (Lab) | FAX +65 6478 9924
      / !! \  michelle_mok at bsf.a-star.edu.sg<mailto:michelle_mok at bsf.a-star.edu.sg>

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