Do you know the hydrophobicity values (Log Kow) of your compounds?

From what you have observed I reckon that the drug A is killing the cells by initiating apoptosis while drug B may be acting by necrosis/senescence. For primary differentiation you first need to first determine the cell cycle changes which may be induced by your drugs (by PI staining-FACS). Once you get an insight you can proceed further. If it is not apoptosis you can go ahead with apoptosis/necrosis differentiation (Annexin V-PI FACS).

even I see this kind of phenomenon, when I treat my cells with the desired drug and positive control. The cells treated with positive control (Methotextrate) remain attached to the culture vessel, although they loose viability after drug treatment whereas the cells treated with desired drug detach from the culture vessel as soon as they loose viability.

i think you should try and look for DNA fragmentation, which indicates apoptosis, as even I did this assay to relieve confusion. In my case both the desired drug and the positive control exhibited DNA ladder like pattern, which indicates apoptosis.

I think you should try DNA Fragmentation.

Live cell imaging is good approach to have an idea on the morphology. next you go for the molecular events involved? first test if it is apoptosis ((caspase activation, DNa degradation, PARP cleavage,...). if not it is a form of regulated necrosis. also here you can test which molecular players are involved (RIPK3, MLKL, PARP1, NOX, CypD, iron metabolism). Have a look in methods review we wrote, this should already help you.

I agree mostly with James Matthews.

Annexin V/PI assay not only tell you whether it is apoptosis or necrosis, but also tells you the propotion of the 2 types of death. From previous experience, I will guess both apoptosis and necrosis will heppen with both of your compounds. The matter is what type is the main pathway of death. DNA ladder or caspases will only tell you whether apoptosis happened, but will not tell you whether it is the main type of cell death.

Once you know which way to go, then you can test a set of molecular regulators, as Tom Vanden Berghe suggested.

Also, you said compound A behaved like doxorubincin. Doxorubincin interacts with DNA and inhibits topoisomerase II. You could also test compound A in that way.

Be carefull with this.

AnnexinV/PI will not discriminate between apoptosis and necrosis unfortunately!

It is generally used, and often wrongly interpreted.

All cells in stress will expose AnnexinV, even autophagic cells. there are other and better ways to discriminate betwen cell death types..

We use Sytox Red for necrosis.

Article Intrinsic cleavage of receptor-interacting protein kinase-1 ...

Since Scott cited me I'd better answer too. There are many more types of cell death than just necrosis or apoptosis, Tom's review sums up quite a few. I find that the use of inhibitors can be very insightful. By definition, apoptosis is caspase dependent, thus a broad-spectrum caspase inhibitor (such as Z-VAD-FMK) will prevent it. Necroptosis is RIPK1 dependent, so necrostatin-1 will prevent (although it has some off-target effects too). In either case it is best to titrate your compounds in the absence or presence of inhibitors. A multiplex assay will also be insightful. If you have a decent fluorescence plate reader, you could combine Ac-DEVD-AFC (or AMC), Sytox Green and Alamar blue to detect caspase activation, cell permeabilization and viability simultaneously.

Using annexin V-FITC and PI you can distinguish four cell populations : 1. live cells, 2. early apoptotic cells, 3. late apoptotic and necrotic cells and 4. damaged cells

Morphological analysis of cells undergoing programmed death is very important !!!

Consider testing cytochrome C release into cell supernatant (compound B) as indicator of plasma membrane damage and into the cytosol (compound A) as indicator of mitochondrial damage without plasma membrane disruption. Several companies sell cytC ELISA.

First please consider that we know about more than 10 types of cell death subroutines (as Bram said) so I think discriminate between apoptosis and necrosis is not the best aim but at least can narrow the possibilities. (Check: Molecular definition of cell death subroutines_CDD_2012)

As you wrote about adherent cells I do NOT recommend you to use vital staining with FACS like the mentioned AnnexinV test. Due to the harvesting step (which is needed for FACS measurement) you may cause an uncontrolled damage on the cell surface antigens (like phosphatidylserine) and on the membrane that gives you false positive results. However there are other ways to detect the AnnexinV positivity (like fluorescent microscopy). Moreover I share the opinion of Tom and Vydia that AnnexinV positivity can be occurred in more cell death types not just in apoptosis.

Focusing on your question, you wanted to know which experiments would help to discriminate between the two cell death types. I would say: which sets of experiments help to characterize your cell death types. :)

First I recommend you to check the literature, Tom and I also suggested you two reviews in the topic. I would also check those papers that focusing on your type of cell line, because this can be crucial. Then I would start with the time course and dose response experiments (in agreement with James) with carefully selected positive controls (like cell death ligands, intrinsic inducers, DNA alkylating agents etc.) as Himanshi recommended you. Check the ladder type DNA fragmentation with SubG1 FACS assay or even with simple agarose gel electrophoresis and with dye exclusion tests, like trypan blue, Alamar blue (like Bram said) or PI positivity (the later can be detected by fluorescent microscopy instead of FACS, see the reason above). I would check maximum 20-24 hrs of incubation time, later you may detect just cell debris (except some cases). But you can check approx 8-12 hrs incubation time too. In the meantime the state of the mitochondria can be also very informative; you can measure the transmembrane potential in time course experiment with imaging techniques. Next step is to check the involved molecular players like Tom and others said. Activity of caspase 8/9/3, RIPK3, Omi/HtrA2, Bcl2 family, PARP1, AIF; cleavage of PARP1, RIPK1; release of cytochrome c, localization of AIF; ATP and NADP/NADPH level etc. And of course I perfectly agree with Lidia about the importance of cell morphology.

Good luck! :)

A cell can die in a number of ways. Apoptosis, necroptosis and autophagy mediated or modulated cell death. How to determine the mechanism of cell death induce by any treatment?

First of all, be aware that MTT is not really a method for determining cell death. MTT measures mitochondrial activity, not necessarily cell death. Trypan blue exclusion is a good, rapid, basic measure of cell death.

So, mode of cell death - apoptosis - zVAD is a pan caspase inhibitor, use of this chemical will inhibit apoptosis and help you to determine whether apoptosis plays a roe in the cell death seen.

Necroptosis - Necrostatin will inhibit necrosis, via modulation of RIPk-1, this will help you to determine whether necrosis plays a role in the cell death seen.

Autophagy - 3MA inhibits autophagy, this will help you to determine whether autophagy plays a role in the cell death seen.

The assessment of cell death can be done using trypan blue.

You got some interesting data on the effects of new substances . Congratulations. Assume that these substances is initiated under certain conditions of cell death different ways . You know that this apoptosis, necrosis , autophagy . You also know that the necrosis often begins with pathological changes in the cell membrane . And then comes a cascade of intracellular processes , some of which are also characteristic of apoptosis. Key mechanisms of apoptosis associated with the processes at the level of the cell nucleus . Thus , the new substance capable initiates processes as from the periphery of the cell ( membrane) , and a cell nucleus (genetic unit ) . So the first thing you must learn in a dose- dependent pathological processes. It is very likely that at high doses will prevail necrotic changes . At low doses will include mechanisms of apoptosis. In the comments you got the advice to use morphological methods for visualizing the characteristic features of apoptotic bodies formation , signs of damage to the membrane of mitochondria. I believe this is the right advice at the initial stage of the analysis . At the next stage it is advisable to attach more costly techniques associated with the analysis of the caspase and other ways of development of cell death . Good luck to you .

I suggest flow cytometric analyses. Annexin V-FITC and propidium iodide staining will give you some evidence about the mode of action of compounds. you can detect the apoptotic cells (early and late) and necrotic cells. Also you can do cell cycle anlayses with flow. You can detect sub G0 as apoptotic cells and cell cycle phase distribution. you may see the cell cycle arrest. good luck.

I agree the answers above me. So I will stick to some extra quite easy tests you could do:

- Caspase 3/7, 8 or 9-GLO assay (Promega). If you do this in you're treated and untreated cells (and I would do it on different time points) you can see if the caspases play a role. When they do, it's most likely apoptosis.

- Mitochondrial depolarisation can be meassured by facs with TMRE

- For autophagy you could look at LC3B/p62 on for example westernblot or immunocytochemistry.

- An other marker for cell death on facs is 7AAD

There are a lot of ways to determine the cell death proces. Just read into them (apoptosis, necrosis, autophagy) and try to determine the specific markers for those processes in you're cells.

I agree the answer above me. The easy one is the Annexin V-FITC and PI staining using flowcytometry. As Lidia wrote you can distinguish four cell populations : live cells, early apoptotic cells, late apoptotic and necrotic cells and damaged cells.

The most rapid assay to distinguish viable, early apoptotic and late apoptotic/necrotic cells is: Annexin A5/PI co-staining and FACS (it takes 20-30 min). Good luck!

I agree with the James Mathews suggesion. If you want to find out the type of cell death you need to do Annexin V/PI. But if you need the compleate pathway then first you have to know the cell cycle phase arrest using flowcytometry and based on the phase arrest you need to find out the up/down regulating signaling check points in that particular cells.

The only thing I can think to add that might be of some help is the use of the Yopro-1/ PI staining. Life technologies has a protocol and a kit for this or you can buy them separately but use their protocol (the cheaper option). I've found it much easier and more reliable than using annexin V, which is also a more specific marker than using Yopro-1 which is a more general stain.

Following this I would then start to investigate the more specific markers such as cytochrome c for intrinsic apoptosis or the various caspases.

Good luck.

I am so happy to have received so many responses on my question. I would like to thank all the contributors for your kind replies. The most important infromation i got from the answers is to perform some experiments to distinguish apoptosis and necrosis. That would be helpful to my research.

You can distinguish cells undergoing apoptosis, necrosis, and autophagy using flow cytometry, and light/fluorescent microscopy methods

Darzynkiewicz et al., Flow cytometric analysis of cell death: A critical review. Cytometry Res. 1995 Vol. 5 , 1-8.

Darżynkiewicz et al., Critical aspects in the analysis of apoptosis and necrosis Hum Cell 1998 Vol. 11, 3-12

Recently, necrosis is considered as programmed cell death

I agree with wonderful tips Lidia Mazur on the methods of detection of apoptosis and necrosis . However, the last sentence of the boards needs to be clarified . It is a question that most of the processes in living cells develop the program selected during evolution. Therefore, a number of stages in the development of apoptosis and necrosis are very similar . But there are key features . Apoptosis begins inside with the implementation process of programmed cell death ( within - outwards ) . Necrosis begins with the cell membrane (from outside - to inside). And then , the process of necrosis can affect the cell nucleus .

During necrosis changes in the cell nucleus occur later than during apoptosis.

In the early stages of apoptosis, chromatin fragmentation and DNA degradation are observed.

However, programmed necrosis can be a continuation of the process of apoptosis.

The ATP level determines the type of death occurring in the cell.

We forget that often see the development process with an intermediate or final stage. And often do not have the right to talk about events in the precise temporal context. Therefore, it is important for young scientists understanding of the direction of the pathological process. Apoptosis - this is from the nucleus to the cell membrane. When necrosis - from the cell membrane - to the nucleus.

Phosphatidylserine externalization is accepted to be the event occurring during early stages of apoptosis.

An easy way to determine if your cells are dying by apoptosis would be to incorporate the pan-caspase inhibitor zVAD into your experiments. This compound effectively suppress apoptosis, whereas alternative kinds of programmed cell death such as necroptosis progress fine. Including the compound Necrostatin-1 could help you determine if your cells die through necroptosis.

You can distinquish apoptotic and necrotic cells under light or fluorescent microscope using May-Grunwald / Giemsa and orange acridine, respectively

In cells undergoing autophagy, the increased autophaphic vesicle number (staining Wright Giemsa or May Grunwald Giemsa / light microscopy) and lysosomal activity (LysoTracker / fluorescence microscopy) can be observed