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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