Re: detecting apoptosis in one cell type in coculture

Posted by mmodel on
URL: http://confocal-microscopy-list.275.s1.nabble.com/detecting-apoptosis-in-one-cell-type-in-coculture-tp7584201p7584213.html

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:

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>
> 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/
>