detecting apoptosis in one cell type in coculture

[Leoncio Vergara-2]

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Good morning

We are working on setting up an image screening assay in which we want to
measure apoptosis in one cell type (adherent)  after exposure to T cells
(non adherent). the adherent cells are grown on a monolayer and the T cells
are added on top and incubated for a few hrs (~3-5 hrs). We want to study
apoptosis in the adherent cells without interference from the T cells.
CellEvent (Thermo Fisher) works great in other conditions and is ideal for
a screening assay, but in this case it does not work because of the
presence of the Tcells, We would like to know is there is an alternative
indicator we can load in the adherent cells and wash prior to the T cells.
We need to test multiple cell lines so fluorescent protein based indicators
would not be ideal.

Thanks in advance for your help.

Leoncio Vergara MD
Co-Director

Center for Advanced Imaging (CAI) at the

Center for Translational Cancer Research (CTCR),

Institute for Biosciences and Technology,

Texas A&M Health Sciences Center,

Houston, Texas 77030

[mmodel]

Hi Leoncio

Apoptosis is usually not decided by a single assay but by a combination of several assays. Besides, apoptotic cells can sometimes detach, depending on the cell type and the stimulus. My first thought is to label T cells with a fluorescent marker (calcein), then trypsinize everything and analyze those that are calcein-negative using, for example, DNA aggregation (blue), depolarization of mitochondria (red) and maybe a far-red assay for caspase. Or if you can use a confocal system then T cells sitting on top shouldn't be an issue

Mike

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Leoncio Vergara
Sent: Wednesday, September 09, 2015 12:31 PM
To: [hidden email]
Subject: detecting apoptosis in one cell type in coculture

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Good morning

We are working on setting up an image screening assay in which we want to measure apoptosis in one cell type (adherent)  after exposure to T cells (non adherent). the adherent cells are grown on a monolayer and the T cells are added on top and incubated for a few hrs (~3-5 hrs). We want to study apoptosis in the adherent cells without interference from the T cells.
CellEvent (Thermo Fisher) works great in other conditions and is ideal for a screening assay, but in this case it does not work because of the presence of the Tcells, We would like to know is there is an alternative indicator we can load in the adherent cells and wash prior to the T cells.
We need to test multiple cell lines so fluorescent protein based indicators would not be ideal.

Thanks in advance for your help.

Leoncio Vergara MD
Co-Director

Center for Advanced Imaging (CAI) at the

Center for Translational Cancer Research (CTCR),

Institute for Biosciences and Technology,

Texas A&M Health Sciences Center,

Houston, Texas 77030

[samuel connell]

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Hi Leo,

One idea would be to load the adherent cells with TMRE or TMRM. These dyes
bind to mitochondria with high mitochondrial transmembrane potential and
then are released from the mitochondria when this dissipates. One would
probably prefer to witness cytochrome C release from the mitochondria, but
this is a more time consuming assay design with GFP cytochrome C. Once you
have these cell lines expressing cleanly, those assays are relatively
straight forward.

There's some other related ideas in a Methods paper I worked on a while
back with some fantastic long term collaborators in Doug Green's lab.

Measuring apoptosis at the single cell level.

Free access is available now:

http://www.ncbi.nlm.nih.gov/pubmed/18314052

Cheers,
--
Sam

On Wed, Sep 9, 2015 at 9:31 AM, Leoncio Vergara <[hidden email]>
wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Good morning
>
> We are working on setting up an image screening assay in which we want to
> measure apoptosis in one cell type (adherent)  after exposure to T cells
> (non adherent). the adherent cells are grown on a monolayer and the T cells
> are added on top and incubated for a few hrs (~3-5 hrs). We want to study
> apoptosis in the adherent cells without interference from the T cells.
> CellEvent (Thermo Fisher) works great in other conditions and is ideal for
> a screening assay, but in this case it does not work because of the
> presence of the Tcells, We would like to know is there is an alternative
> indicator we can load in the adherent cells and wash prior to the T cells.
> We need to test multiple cell lines so fluorescent protein based indicators
> would not be ideal.
>
> Thanks in advance for your help.
>
> Leoncio Vergara MD
> Co-Director
>
> Center for Advanced Imaging (CAI) at the
>
> Center for Translational Cancer Research (CTCR),
>
> Institute for Biosciences and Technology,
>
> Texas A&M Health Sciences Center,
>
> Houston, Texas 77030
>

[Markus Rehm]

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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/

[Leoncio Vergara-2]

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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:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on 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/
>

[Markus Rehm]

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*****

Thanks for the additional info. In my opinion, this won't be easy to do
given the imaging modalities and requirements you describe. Rather than
spending a lot of time on establishing workflows and readouts that in the
end may not be reliable, it could be an option to break it down a bit.

Would a simple initial analysis based on a robust live/dead readout such as
propidium iodide or cytox variants be an acceptable first step? Cheap,
simple, strong signal change, easy to analyse. Once you have identified the
conditions that result in high amounts of cell death you could investigate
those conditions separately at higher detail.
When you fix your cells it should be rather straight forward to go in
straight away and have a look at higher resolution to see if you can get a
first idea of whether the T cells, adherent cells or both are dying in wells
that are of interest to you.

A recommendations for the second round would then be to include controls in
which you add a caspase inhibitor such as zVAD-fmk or QVD-oph. If cell death
drops, you have a good indication that it is apoptotic cell death. You may
be able to label your T cells either based on a specific surface marker (AB
staining) or a dye that binds to cytosolic structures/proteins (don't label
with dyes that remain soluble, since they will be lost from the cells once
they die) to separate these from the rest.

If you wish to do time lapse analysis of single cells I recommend to avoid
(short wavelength) DNA binding dyes as much as possible (they would normally
be used for tracking). Their general toxicity and the phototoxicity in most
cases potentiates the apoptosis responses and your results may not be
representative. Typically one would find much higher amounts of death in the
field of view than outside the field(s) of view used for timelapse analysis.

The suggested AnnexinV staining on living cells for time lapse imaging is
not so straightforward, since the binding buffer that normally needs to be
used to get good staining cotains high conc. of calcium (if I remember
correctly) and your cells may not like such an overload at all.

best
Markus

[mmodel]

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:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on 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/
>

[samuel connell]

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*****

Hello again Leo,

CMXRos, or Mitotracker Red is a fixable mitochodrial dye. It's loading is
dependent on membrane potential. This should provide a high signal in
intact mitochondria, and would become quite low in apoptotic cells.

--
Sam

On Thu, Sep 10, 2015 at 1:12 PM, MODEL, MICHAEL <[hidden email]> wrote:

> 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
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> 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:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > Post images on 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/
> >
>

[Alfred Bahnson]

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*****

Leoncio,

I would encourage you to pursue an approach based on time-lapse imaging and
automated image analysis to detect the transient episodes of death of
individual cells among your adherent target cell population over time.  A
time-lapse approach is important because in many cases, the adherent cells
phagocytose the killed cells shortly after death.  You will see this in the
"videos."  End-point assays only tell part of the story; they miss the
journey that your culture took to get there.

You can readily detect cell death by uptake of propidium iodide (PI) in
your TRITC channel.  The excitation wavelength is less photo-toxic than
with the FITC and DAPI channels, and exposures can be minimized in time and
intensity because the PI signal is strong.

I would recommend setting up grids where you acquire sets of adjacent
viewfields that can be stitched together after acquisition so that you have
sufficient cell numbers for good statistics.  Run duplicate wells and
duplicate grids within wells to estimate precision.  Working in multi-well
plates with 15 minute scan intervals, you will have time to include a lot
of viewfields.

Development of the automated image analysis method will be a challenge
requiring the skills of at least one expert or dedicated person willing to
become an expert.  I don't think there is yet any assembled package that
can be plugged in for this purpose (I'd like to hear otherwise!), but there
are many places to start: ImageJ (FIJI), BioImageXD, and CellProfiler are
free and open-source platforms that encourage users to share in
development.  I think I've said that correctly, more or less.  We have used
these programs successfully for various projects without ever altering the
"source" code.

An attractive feature of the automated image analysis approach is that the
method can evolve to become more efficient and more automated over time,
and as improvements are made they can be tested on past image sets,
effectively improving previous data (no harm in that, right?).  So right
off the bat it is important to emphasize and seek consistent quality in the
image set: focus, exposure, contrast adjustment for bright-field or phase.
Using a small z-stack can be helpful.  Phase is a problem in multi-well
plates.  I am mentioning non-fluorescent imaging parameters because your
analysis method will probably need to incorporate some logical approach for
discerning T-cells from target cells, and you will likely need to put
together features from either phase-contrast or bright field images to help
will this, in addition to the PI signal from your fluorescent images.  You
will also probably want to count live cell numbers in each viewfield for
purposes of having a denominator upon which to quantify death rates.
However, it is possible that the size of your dying cells alone, based on
fluorescence, could distinguish between T-cell death and target cell death
in many cases.

A lot depends on where you are now in terms of familiarity with methods for
segmentation (FIJI's "Trainable Weka Segmentation is very cool:
http://fiji.sc/Trainable_Weka_Segmentation) and putting together steps in
an automated method for outputting counts from large image sets.  Also,
when it comes to optimizing non-fluorescent imaging of cells for automated
segmentation (for live-cell counts and possibly for distinguishing target
cells and T-cells), it's not easy.  I would like to see a table of the many
approaches to this goal, the pros and cons of each method.  Unfortunately,
each different type of target cell may require a somewhat different
solution.  Don't expect perfection.  I think attention should be paid to
statistical quality control (based on duplicate wells and duplicate grids
within wells), as well as manual verification of segmentation in
intermediate steps and manual verification of output numbers in randomly
selected image sets.  So the automated analysis method needs to incorporate
manual verification at various stages for quality control / method
validation.

Of course, if suitable fluorescent probes can be incorporated into this
approach, all the better!

Al Bahnson, Partner
Kairos Instruments, LLC
Pittsburgh PA

412-735-9983













On Thu, Sep 10, 2015 at 11:31 AM, Leoncio Vergara <[hidden email]>
wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> 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:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > Post images on 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/
> >
>

[mmodel]

I only would like to add to that that apoptotic cells may not pick up some of live/dead stains, such as PI, because their membranes remain more or less intact for some time.

Mike Model

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Alfred Bahnson
Sent: Friday, September 11, 2015 9:48 AM
To: [hidden email]
Subject: Re: detecting apoptosis in one cell type in coculture

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Leoncio,

I would encourage you to pursue an approach based on time-lapse imaging and automated image analysis to detect the transient episodes of death of individual cells among your adherent target cell population over time.  A time-lapse approach is important because in many cases, the adherent cells phagocytose the killed cells shortly after death.  You will see this in the "videos."  End-point assays only tell part of the story; they miss the journey that your culture took to get there.

You can readily detect cell death by uptake of propidium iodide (PI) in your TRITC channel.  The excitation wavelength is less photo-toxic than with the FITC and DAPI channels, and exposures can be minimized in time and intensity because the PI signal is strong.

I would recommend setting up grids where you acquire sets of adjacent viewfields that can be stitched together after acquisition so that you have sufficient cell numbers for good statistics.  Run duplicate wells and duplicate grids within wells to estimate precision.  Working in multi-well plates with 15 minute scan intervals, you will have time to include a lot of viewfields.

Development of the automated image analysis method will be a challenge requiring the skills of at least one expert or dedicated person willing to become an expert.  I don't think there is yet any assembled package that can be plugged in for this purpose (I'd like to hear otherwise!), but there are many places to start: ImageJ (FIJI), BioImageXD, and CellProfiler are free and open-source platforms that encourage users to share in development.  I think I've said that correctly, more or less.  We have used these programs successfully for various projects without ever altering the "source" code.

An attractive feature of the automated image analysis approach is that the method can evolve to become more efficient and more automated over time, and as improvements are made they can be tested on past image sets, effectively improving previous data (no harm in that, right?).  So right off the bat it is important to emphasize and seek consistent quality in the image set: focus, exposure, contrast adjustment for bright-field or phase.
Using a small z-stack can be helpful.  Phase is a problem in multi-well plates.  I am mentioning non-fluorescent imaging parameters because your analysis method will probably need to incorporate some logical approach for discerning T-cells from target cells, and you will likely need to put together features from either phase-contrast or bright field images to help will this, in addition to the PI signal from your fluorescent images.  You will also probably want to count live cell numbers in each viewfield for purposes of having a denominator upon which to quantify death rates.
However, it is possible that the size of your dying cells alone, based on fluorescence, could distinguish between T-cell death and target cell death in many cases.

A lot depends on where you are now in terms of familiarity with methods for segmentation (FIJI's "Trainable Weka Segmentation is very cool:
http://fiji.sc/Trainable_Weka_Segmentation) and putting together steps in an automated method for outputting counts from large image sets.  Also, when it comes to optimizing non-fluorescent imaging of cells for automated segmentation (for live-cell counts and possibly for distinguishing target cells and T-cells), it's not easy.  I would like to see a table of the many approaches to this goal, the pros and cons of each method.  Unfortunately, each different type of target cell may require a somewhat different solution.  Don't expect perfection.  I think attention should be paid to statistical quality control (based on duplicate wells and duplicate grids within wells), as well as manual verification of segmentation in intermediate steps and manual verification of output numbers in randomly selected image sets.  So the automated analysis method needs to incorporate manual verification at various stages for quality control / method validation.

Of course, if suitable fluorescent probes can be incorporated into this approach, all the better!

Al Bahnson, Partner
Kairos Instruments, LLC
Pittsburgh PA

412-735-9983













On Thu, Sep 10, 2015 at 11:31 AM, Leoncio Vergara <[hidden email]>
wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> 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:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > Post images on 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/
> >
>