I'm struggling to see the benefits of TUNEL/AnnexinV/Caspase. As far as I can tell, Annexin can pick up early and late stage apoptosis, but even with the addition of a nuclear dye (e.g. PI) its difficult to distinguish late stage apoptosis from necrosis. Caspase assays can show early apoptosis and TUNEL late stage (but again difficult to distinguish late apoptosis from necrosis).
Is there a compromise between these assays that allow the detection of apoptosis over necrosis?
Thanks for your help!
john
There are strength and weaknesses for all type of apoptosis methods. I have attached two screen captures of the same treatment one measured with TUNEL and measured annexin V/caspase. If you add up the apoptotic cells the caspase method would give you a total of 45% versus TUNEL 36%. Whereas the TUNEL gives 64% non apoptotic and caspase give 54.5% non apoptotic. So what you should conclude here is that your data will be much stronger if you measure apoptotic cells using more than one method.
You can have apoptosis and necrosis occurring at the same time in the same set of cells and also senescent cells. Quick and dirty necrosis measurement is measuring lactate dehydrogenase release into culture media this can be used to confirm your staining results.
TUNEL is specific for apoptotic cells and annexinV/caspase/PI can measure alive/dead/mid/late apoptotic while also giving you an idea of the dead cells which are likely necrotic cells.
Good luck
Screen capture credit Dr. Ruan
I totally agree with David. This is especially true if you have the intention to publish your paper. I have experienced reviewers having totally different opinion about one assay is more adequate than the other. Not uncommonly I will be asked to do a specific assay simply because the reviewer's personal preference. I will say at least two assay so that yourself feel comfortable that the change is real.
I totally agree with David. But as for as your question regarding necrosis versus apoptosis is concerned, It is quite possible to figure out the proportionate differences at early apoptotic stages labeling samples/ comparing samples with nuclear staining dyes (Propidium Iodide) and surface marker dyes (Annexin V). During late apoptotic stages it's become more difficult as most of the apoptotic cells lose their membrane integrity thus become porous and will give a false signal for propidium Iodide staining. That's why referees and people around the world ask for/agree for to use two or three different methods of validating the percentage of apoptosis. But still ideologically, necrotic cells will not stain with annexin and specific caspases in downstream of apoptotic pathways.
Hello.
You can also use some new method such as Multimodal Holographic Microscopy.
See link
https://www.researchgate.net/publication/274088124_Multimodal_Holographic_Microscopy_Distinction_between_Apoptosis_and_Oncosis
Article Multimodal Holographic Microscopy: Distinction between Apopt...
Thanks for the information guys, looks like the best thing to do is use two apoptosis test simultaneously. I like Jorn's idea of using a necrosis inhibitor, has anyone else tried this?
Thanks again!
I just would like to add that you have to be careful with interpreting results from cell death inhibitors. It is often observed that cell death modalities can switch from one to another without an remarkable change in the cell death kinetics. The pan-caspase inhibitor zVAD-fmk for example can force apoptotic cells into necroptosis. It may therefore look like that all your cells are necrotic (because the apoptosis inhibitor didn't protect), but zVAD actually shifted your apoptotic cells into necroptosis. In addition, the RIPK1 kinase inhibitor necrostatin-1 can, despite its name, also protect against certain forms of apoptosis. The best way is indeed to combine several cell death detection assays. Although not quantative, studying the morphological features of your dying cells can already tell you a lot.
I think that if one follows the "standards" detailed in the attached reviews, it will be difficult to criticize him / her ...
Best regards
Robert
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