Dr. Jian She asked for comments on her observations of the presence of cells
that were annexin V-/caspase-3+. .
It is likely the cells that are caspase 3 +/Annexin V - are early
apoptotic cells that have already activated caspase(s) but yet did not
exposed phosphatidylserine on the outside leaflet of the plasma membrane.
Apoptosis is a kinetic process and the "time-window" through which we see
this process depends very much on the marker (assay) that is being used
(e.g. see chapter 24 Methods in Cell Biology; Vol 63, 2001) ). Some assays
(e.g. TUNEL) detect late apoptotic cells because DNA degradation is a late
event, subsequent to activation of caspases. In contrast, activation of
caspases is one of the early events. Thus, apoptotic index of the same cell
population estimated by different assays may vary. Dr. She by gating out the
cells with low scatter values selectively removed the late apoptostic
(and/or necrotic) cells ("nacrotic stage" of apoptosis) which were annexin
V+/PI+ and annexin V+/caspase-3+ (51% before gating, 34% after the gating;
this population also seems to spill to annexin V+/caspase-3- quadrant).The
annexin V-/caspase-3+ cells, thus, appear to be apoptotic cells at early
stage of apoptosis, with the plasma membrane integrity still preserved.
However, the situation presented by Dr. She is complicated by a possibility
that necrotic cells may also be present. . The concentration of CPT (4 uM)
used to induce apoptosis was extremely high and could also induce cell
necrosis. Necrotic cells, however, are expected to be annexin V+/PI+/low
scatter.. .
Zbigniew Darzynkiewicz
Brander Cancer Research Institute
New York Medical College
19 Bradhurst Ave.
Hawthorne, NY 10532
tel: 914-347-2801
fax: 914-347-2804
http://www.geocities.com/z_darzynkiewicz
> -----Original Message-----
> From: Jian She [SMTP:Shej@war.wyeth.com]
> Sent: Tuesday, May 08, 2001 4:15 PM
> To: Cytometry Mailing List
> Subject: Re: About double positive population of Annexin V and
> Caspase-3
>
> Dear Dr. Darzynkievicz or other flow experts:
>
> In my experiment for Apoptosis, I have a difficulty to understand
> population (Caspase-3
> "+" and Annexin V "-" cells. Could you help me to explain this
> population?!! Attached is the FACS analysis results.
>
> Thanks;
>
> Jian She
>
> >>> <mgumanovskaya@pharmingen.com> 05/08 2:09 PM >>>
>
> Dear Dr. She,
> Thank you for your interest in PharMingen's and Transduction Laboratories'
> reagents. I am having a hard time to explain why are you getting
> Caspase-3
> "+" and Annexin "-" cells . I am going to pick other peoples brain :) ,
> but
> I would also recommend for you to send an e-mail to flow cytomerty mailing
> group. It is great way to people like Dr. Darzynkievicz and other to help
> with your question. You can address it: Dear flow experts :) or something
> like that. Many flow users read this every day and chances are somebody
> had
> same issue.
>
> Email address is : cyto-request@flowcyt.cyto.purdue.edu
>
> Best regards,
>
> Dr. Marina Gumanovskaya
> Technical Service
> BD Biosciences
> PharMingen and Transduction Laboratories
>
> "Jian She" <Shej@war.wyeth.com> on 05/02/2001 02:43:39 PM
> To: Marina Gumanovskaya/SDCA/BDX@BDX
> cc:
> Subject: About double positive population of Annexin V and Caspase-3
>
> Please focus on:
>
> The phenotype of Annexin V negative and Caspase-3 positive population.
>
> What is biological mean?
>
> Jian She, M.S, M.D.
> Senior Research Scientist I
> Wyeth-Lederle Vaccines
> Bldg. 190/Rm. 4118
> 401 N. Middletown Rd.
> Pearl River, NY 10965
> Phone: 845-732-4836
> Fax: 845-732-3628
> SHEJ@war.wyeth.com
> << File: Cell Gated.ppt >> << File: Caspase+Annexin V-2.ppt >> << File:
> Caspase-3.ppt >> << File: Caspase+Annexin V-1.ppt >>
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