Posted by
Markus Rehm on
URL: http://confocal-microscopy-list.275.s1.nabble.com/detecting-apoptosis-in-one-cell-type-in-coculture-tp7584201p7584212.html
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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