>>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