http://confocal-microscopy-list.275.s1.nabble.com/detecting-apoptosis-in-one-cell-type-in-coculture-tp7584201p7584214.html
CMXRos, or Mitotracker Red is a fixable mitochodrial dye. It's loading is
dependent on membrane potential. This should provide a high signal in
> Chromatin aggregation is a very specific apoptotic marker. Nuclei split
> into bright dots, so I think Hoechst (but not DAPI) would an obvious
> choice. Some caspase-3 substrates can be pre-loaded into cells. They can be
> expensive but that's also a specific marker. Not sure what else you can put
> into cells before the assay that would withstand fixation. Apoptotic cells
> have altered shapes but how exactly - that may differ from one cell type to
> another.
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:
[hidden email]]
> On Behalf Of Leoncio Vergara
> Sent: Thursday, September 10, 2015 11:31 AM
> To:
[hidden email]
> Subject: Re: detecting apoptosis in one cell type in coculture
>
> *****
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>
> Thanks for the responses so far. I am getting some interesting leads to
> follow but I am realizing the solution may not be straightforward. I have
> quite a bit of experience in several forms of optical microscopy but I am
> new apoptosis. I am quite excited with my involvement in image based high
> throughput screening for cancer research.
>
> We are trying to develop an image based assay moving from flow cytometry
> to imaging. Our first approach was to modify the Flow assay already in
> place for use in imaging and it was then that we found this new set of
> problems.
> Our goal is to setup an assay on fixed cells in large format multiwell
> plates. In the process we plan to do live cell imaging experiments our
> final goal is to setup a drug screening protocol.
>
> I was hoping to find a marker ( or combination of markers) to quantify
> cell toxicity which can be loaded into cells and then washed before adding
> T cells to the cultures. Non wash assays like CellEvent and Annexin V have
> the problem of interference from the Tcells. An option is to label the
> Tcells to separate them on analysis, but at high seeding ratios the Tcells
> cover most of the well area and at the low magnifications (and hence low
> NA) used for screening we don't have enough z-discrimination to separate
> the Tcells on top from the tumor cells underneath. We have 4 channels
> available, the classical DAPI, FITC, TRITC and Cy5 combination (405,
> 488,561 and 640 excitations).
>
> One of the goals is to work with multiple cell lines (more than a dozen)
> so any method based on expressing FP based indicators would make the assay
> too complicated.
>
> Leoncio
>
>
>
> On Thu, Sep 10, 2015 at 2:41 AM, Markus Rehm <
[hidden email]> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> >
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http://www.imgur.com and include the link in your
> posting.
> > *****
> >
> > Dear Leoncio,
> >
> > Could you expand a bit on what your requirements for the experiment are?
> > Do you need time-lapse information of apoptosis kinetics and cell
> > tracking or are end point read outs sufficient? Depending on your
> > needs, the approach taken may be quite different.
> >
> > Best wishes
> > Markus
> >
> > Dr. rer. nat. Markus Rehm
> > Biomedical Research Lecturer
> > Dept. of Physiology & Medical Physics
> > & Centre for Systems Medicine
> > Royal College of Surgeons in Ireland
> > RCSI York House
> > York Street
> > Dublin 2
> > Ireland
> >
> > phone: 00353 (0)1 4028563
> > email:
[hidden email]
> >
https://research1.rcsi.ie/pi/mrehm/> >
http://www.systemsmedicineireland.ie/> >
>