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>>I have recently been attempting Annexin V staining and so far this
>>has been succesful. However I am worried about the amount of debris in t=
he
>
>>bottom left of FSC/SSC, should this be included in the final analysis
>>or excluded?
>>
>I guess the answer depends on whether the events w/ low FS ansd SS are
>apoptotic cells or debris or some other cell type [depending on the system=
].
> For th e most part, the info you want is % apoptotic cells of a particula=
r
>population. So you need some method to determine whole cells [apoptotic o=
r
>live] from other events. If you can't achieve this with LS then maybe
>adding a McAb or staining for DNA wil l be a required gating step.
>
>Good luck,
>Tom
Hi James,
To add to Tom's answer, the events with low SSC and low FSC may depend on=
whether you have FSC and SSC amps set to log. If this is so then what you a=
re
really seeing is debris form your sheath fluid. You could try a linear scal=
e for
the FSC if this is the case.
Tom suggests that you use an antibody to detect your cell population, if t=
he
scattering is cellular debris then your Ab may stick to it, giving you a fa=
lse
positive. A DNA stain is more useful. If you are doing apoptosis work then =
you
may want to discriminate the necrotic cells from the apoptotic cells. Most=
people use PI to do just that.
Andy Riddell
PNAC Division
MRC Laboratory of Molecular Biology,
Hills Road
Cambridge CB2 2QH
U.K.
tel: (01223) 402 218
fax: (01223) 412 178
e-mail: ar3@mrc-lmb.cam.ac.uk
=05=05=05 Andy Riddell
PNAC Division
MRC Laboratory of Molecular Biology,
Hills Road
Cambridge CB2 2QH
U.K.
tel: (01223) 402 218
fax: (01223) 412 178
e-mail: ar3@mrc-lmb.cam.ac.uk
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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;
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