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I've one question about annexin V staining and its possible influence &
effect on scatter characteristics of cells.
I try to determine the amount of apoptotic cells by annexin V staining kit
(PharMingen) and subsequent analysis on a FACScan. The conditions used in
my experiments comprise incubation with apoptosis inducing antibody CH11
(Immunotech) and a variety of cytokines e.g. TNF-alpha. The cells I use for
my investigations are human leukemic B-cells (mature B immunotype).
On analysis of the cells, I experience a change in scatter characteristics
(see ASCII depiction below). All prior experiments (e.g. CD95 expression,
bcl-2 expression) showed a typical scatter picture of lymphocytes or
lymphoblasts with a medium forward scatter and a low side scatter. Staining
with annexin V causes the cells to have a low forward scatter and a medium
side scatter so as the dot-plot had been mirrored. This phenomenon occurs
on all conditions analysed (including untreated control). I checked for
possible staining procedure errors, but I didn't find anything to have gone
wrong according to the kit's description and procedure manual.
Has anyone else on the list experienced such a change in scatter
characteristics ? Could this scatter change be due to the binding buffer
necessary for annexin staining (my suspicion, but unproven yet) ?
Any help on this problem is greatly appreciated. Thanks in advance
Best regards,
Symbolic picture of normal scatter Symbolic picture of changed scatter
S ! S !
I ! I !
D ! D !
E ! E ! *
! ! ***
S ! S ! ****
C ! C ! *****
A ! A ! ******
T ! *********** T ! *******
T ! ********** T ! ******
E !--------------------------> E !----------------------------->
R FORWARD SCATTER (FSC) R FORWARD SCATTER (FSC)
If anyone needs more precise data on this problem, I can mail the data files.
Thanks again
_________________________________________________________
Andreas Christaras
Heinrich-Heine University Children's Hospital Duesseldorf
Lab. of Experimental Hematology (KMT-Labor)
Bldg. 14.82 (Gebaeude 14.82) - Moorenstrasse 5
4 0 2 2 5 D U E S S E L D O R F - G E R M A N Y
---------------------------------------------------------
Fon: +49-211-81-16103 Fax: +49-211-81-16191
E-Mail: christar@uni-duesseldorf.de
_________________________________________________________
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CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the
Purdue University Cytometry Laboratories and distributed free of charge
as an educational service to the cytometry community.
If you have any comments please direct them to
Dr. J. Paul Robinson, Professor & Director,
PUCL, Purdue University, West Lafayette, IN 47907.
Phone: (765)-494-0757;
FAX(765) 494-0517;
Web