Mojgan,

Since you are likely to measure the level of apoptosis in the target cells, there are two good ways of doing it:

1. Classical Annexin V/PI staining (the Sigma kit is quite good);

2. Any of the mitochondrial dyes - such as DiOC6 - for the measurement of the changes in mitochondrial membrane potential - Molecular probes.

Both will require flow cytomtery.

If there is a problem of separating effector and target cells by FC, I would recommend labelling the targets with a Fluorescent Linker (Sigma) - easy and works very well (it labels the membranes). However, you won't be able to use Red linker with Ann/PI kit since RFL and PI are both detected through FL2 channel.

You might not be able to pre-label the targets through a specific CD receptor, since these might be affected by membrane changes in apoptosis.

Hope this helps. We have published a paper on a similar subject some time ago which can be found at: http://www.haematologica.org/content/89/4/435

Hi Mojgan,

The following resources might be helpful. One of them has a list of publications which might be relevant. 

Good luck ! 

Thanks Arpan De.

Promega has a kit to determine cell viability. I think its called Cell titer glo. It works well.

Hi,

ّFor adherence cells You can use XTT Cell Viability Assay Kit  that dose not need to DMSO for  solving formazan.... I suggest  that...

If I understand correctly, your problem is that you can't separate the cells from each other, and are worried about the contribution of the lymphocytes to the viability signal?  Perhaps surprisingly there's not a superb answer to that.  

Most classically, this is done by pre-labeling the cancers with 51Cr.  But these days, radioactivity is not for the faint of heart.  

We tried pre-labeling with Calcein-AM, and it works so-so.  There's leakage, and the signal fades over time.  48 hours might just work, but that's pushing it. 

We also used Calcein-AM on mixed populations, at the end of the killing assay.  It turns out that the signal from the lymphocytes is rather low, compared to the cancers, due to their much smaller cell volume.  You can account for it mathematically, or just neglect it. It won't be more than a 10% error, unless your E/T ratio is extreme. 

It's possible to try to use a wash step to remove the loosely adherent lymphocytes while leaving the adherent cancers there, but you have to be extremely gentle, and do it in a way that's somehow automated to achieve consistency. 

Finally, you could do the "poor man's flow cytometer" -- trypsinize everything, put them into a hemacytometer and count by fluorescence microscopy.  You can identify the cancers by size pretty easily. 

One elegant, but very pricey solution is the Xcelligence machine.  It works by measuring current through the bottom of a conductive microplate.  The current is blocked by adherent cells, but pretty much not at all by suspension cells just touching the surface. 

Highly interesting question. Not sure of any simple answer. Hopefully someone with a dedicated SOP will emerge. Physical separation might be an option but you don't say how the mixed cells population came about in the first place (?). Why not test them separately in the first instance? Could a kinetic analysis (protracted) lead to a separate response? Worth checking out the following https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3210817/

But if you are simply wanting to review available methods - then in addition to many great suggestions by colleagues above you can also consider the following discussion  https://www.researchgate.net/publication/221712024_An_overview_of_colorimetric_assay_methods_used_to_assess_survival_or_proliferation_of_mammalian_cells?enrichId=rgreq-d6e35eac86c3110f6d6581382aec0b12-XXX&enrichSource=Y292ZXJQYWdlOzIyMTcxMjAyNDtBUzoxMDM5NzY0OTgzNjg1MTlAMTQwMTgwMTMzNzg3MA%3D%3D&el=1_x_2

I am keen to follow the eventual resolution of this issue

Best W, All.

Article An overview of colorimetric assay methods used to assess sur...

Mojgan,

Since you are likely to measure the level of apoptosis in the target cells, there are two good ways of doing it:

1. Classical Annexin V/PI staining (the Sigma kit is quite good);

2. Any of the mitochondrial dyes - such as DiOC6 - for the measurement of the changes in mitochondrial membrane potential - Molecular probes.

Both will require flow cytomtery.

If there is a problem of separating effector and target cells by FC, I would recommend labelling the targets with a Fluorescent Linker (Sigma) - easy and works very well (it labels the membranes). However, you won't be able to use Red linker with Ann/PI kit since RFL and PI are both detected through FL2 channel.

You might not be able to pre-label the targets through a specific CD receptor, since these might be affected by membrane changes in apoptosis.

Hope this helps. We have published a paper on a similar subject some time ago which can be found at: http://www.haematologica.org/content/89/4/435

I am sorry, only now the second part of your question appeared in my window.

Why is it that you can not do FACS? The FC is not available? It is relatively easy to get the adherent cells off the plastic.

Alternatively - try to use confocal microscope and assess Ann/PI staining after the reaction. In this case you can identify targets easily morphologically.

The easiest (but not a fancy) way of counting dead cells under any microscope would be a simple and straightforward trypan blue stain - only dead cells (but not necessarily apoptotic) will be stained blue.

Could you use an LDH release assay?  This should get around the issue having a mix of target and effector cells.  Promega supply a kit, I have never used it but I know people that have said it works for them.  I am not sure of how much background you would get from the lymphocytes dying off over time but hopefully this should at least be fairly consistent within a single assay.

https://www.promega.co.uk/resources/protocols/technical-bulletins/0/cytotox-96-non-radioactive-cytotoxicity-assay-protocol/

Thanks all. 

**John Schloendorn, yes that is the problem I can't separate them and it is really hard to separate them. 

**Richard K Owusu-Apenten,  the separation is not easy.

**Nina Porakishvili, I can't do FACS actually and no one can't help me, that is the problem, but looking at 1000 cancer cells is easy under microscope?