Normally, it was believed that PI + / Annexin V - cells were necrotic, whereas late apoptosis was double positive for both Annexin V and PI. This allowed for the two different states to be identified.

Now there has been some literature to say necrotic cells start out double positive before they become PI + only. This phenomenon may be cell-type specific. You can use a specific marker of necrosis, necrostatin-1, to identify this population, if you want to consider necrosis vs apoptosis. This will allow you to use the drug at the concentration and length of treatment you want to use.

Please check out this paper: Discrimination between primary necrosis and apoptosis by necrostatin-1 in Annexin V-positive/propidium iodide-negative cells.

Normally both necrotic cells and late apoptotic cells are shown Annexin V + / PI +, so we can differentiate by using lower concentration or lower time exposure for the agent that induce these effects, if it cause Annexin V + with  negative PI (early apoptosis) that's mean this agent induce apoptosis rather than necrosis. 

Thank you!

Normally, it was believed that PI + / Annexin V - cells were necrotic, whereas late apoptosis was double positive for both Annexin V and PI. This allowed for the two different states to be identified.

Now there has been some literature to say necrotic cells start out double positive before they become PI + only. This phenomenon may be cell-type specific. You can use a specific marker of necrosis, necrostatin-1, to identify this population, if you want to consider necrosis vs apoptosis. This will allow you to use the drug at the concentration and length of treatment you want to use.

Please check out this paper: Discrimination between primary necrosis and apoptosis by necrostatin-1 in Annexin V-positive/propidium iodide-negative cells.

Thanks! I'll surely check!

Additionally, PI + / Annexin V won't tell you the death pathway, of which several have been described recently, so activated caspase staining can inform you a lot. Caspase 1 vs. caspase 8 and 9 can tell you about caspase-mediated apoptosis or other programmed cell death pathways (like pyroptosis).

Annexin V + / PI + cells should be considered as late apoptotic or secondary necrotic cells. When it comes to the necrosis, the discrimination between primary necrosis (classical or pathological cell death) or secondary necrosis is essential. In vitro, secondary necrosis follows apoptosis because the cells eventually (inevitably) undergo necrotic cell death (heavy damage to cell membrane) due to the lack of phagocytosis of them by macrophages or other cells (absent in vitro).  That is why it is also called late apoptosis. So, you need to see a shift from annexin V + / PI - field (right bottom area) to annexin V + / PI + area (right upper area) over time if they are truely apoptotic. If they are primary necrotic cells (no apoptosis at all), they rather get populated in the upper left area (annexin V - / PI + area) (and some right upper area as well?). However, this is still a dynamic field because the term of necroptosis has recently been emerged and this is also called programmed necrotic cell death. In fact, this is possibly primary necrotic cell death itself. Anyway. In this case, it may be possible to see the cells directly gathering in the upper right area (annexin + / PI + area) without seeing any shift over time as described above. If you suspect necroptosis, then you had better to use necrostatin (+ your toxic agent). Following necrostatin use, the cell death must be recovered (partially or completely). As a last saying, of course, using just only flow cytometry is not enough for clear definition of cell death. Morphology is always essential to say "apoptosis or other types". In fact, apoptosis is a morphological definition, not a molecular definition, meaning that you need to see the cell morphology for definite decision. Look for pyknosis and/or nuclear fragmentation for apoptosis!. Also, remember that you can see different cell death modalities depending on the dose applied!. Good luck!  

Dear Luis Felipe , in addition to the very good suggestions from colleagues,  I would like to suggest a pper that maybe can help in understanding the dinamic of PI staining, considering also the difficulties that you can experience dealing with adherent cells

Good luck

Cytometry. 2001 May 1;44(1):57-64.

Supravital exposure to propidium iodide identifies apoptosis on adherent cells.

Zamai L et al.

Dr. Enrique Espinosa and Dr. Engin Ulukaya,

You proposed interesting points about cell death, since I had suspected of necroptosis. I've been doing some PCR arrray experiments related to apoptotic pathways with different treatments (drug concentration, 6-hours and 24-hours treatments) to be a bit more sure it's apoptosis. Of course, I'm really considering doing the procedures you suggested.

Dr. Filippo Reno',

Thanks for suggesting me this paper! I'll read it!!

do not rely on a single method. It is not sufficient to discriminate a mechanism as the other before me suggested. There are many other kits available on the market to confirm apoptosis.

I trust only the Annexin V+ /PI- cells. The cells positive to PI in phisiological conditions are just artifacts of the method.

I trust that Annexin V+/PI- indicates early apoptosis while Annexin+/PI+ indicates necrosis. In early apoptosis, the plasma membrane is still intact while in early necrosis the membrane integrity is lost, thus the principle of using FITCH-labelled Annexin V, simultaneously with dye exclusion of PI becomes justified. In this case, intact cells will be FITCH-Annexin V- and PI- because Phosphatidylserine did not translocate to cell surface and the dye excluded. In early apoptosis, there was membrane alteration that could enable Phosphatidylserine externalization to cell surface which enables FITCH-Annexin V binding, but PI exclusion, hence FITCH-Annexin V+ /PI-. In early Necrosis, phosphatidylserine externalization occurs and dye absorbed giving rise to FITCH-Annexin V + and PI+. Thus cells that are FITCH-Annexin V+ and PI+ are on their late apoptosis and may indicate early necrosis as well but not early apoptosis.

Apart from Phosphatidylserine translocation - using FITC conjugated Annexin V peptide,

depolarization of the mitochondrial membrane - using JC-1 dye and Activation of Caspase 3/7 - using FAM-conjugated DEVDFMK peptide could also be applied.

Thank you Michael Ezeani,

I'm glad this topic has raised a great discussion!

The cells pass through several stages,so you should make a kinetics cells death. First the cells will mark only withAnex V and gradually turn to be positive for both markers AnexV and PI(late apoptosis). Maybe you can see different form to cells death in the same population, some cells can pass directly for necrosis cells death PI+ (importantly care with the manipulation: in the case of leukemic cells you can have a 20% of background). To confirm if you’re in present of an apoptosis process you can probe the caspase activation and anti PARP western blot.

Apoptosis conformation needed at least two three prove in diffrent techniques

Thank you very much!

I will regurgitate what every one said along with my observations during experimentation. 

AnxV +ve/PI -ve = Apoptosis

AnxV -ve/PI +ve = Necrosis

However, with flow cytometry it is necessary to determine your correct gate values for Anx+ve cells. A necrotic cell will also be AnxV +ve but to a lesser amount as AnxV can freely diffuse in to a swollen cell and bind to phosphatidylserine. Compared to this an apoptotic cell will have bright membrane AnxV fluorescence. However there will be scenarios where AnxV fluorescence on few apoptotic cells will not be comparable to other brightly AnxV fluorescent apoptotic cells. If you were to observe these cells on flow you may exclude them depending on the gating conditions. 

When you look at Propodium Iodide this can be tricky. All necrotic cells will have brightly stained nuclei but an apoptotic cells undergoing secondary necrosis will some times stain PI positive and some times not. During apoptosis the nuclei will undergo condensation and fragmentation and upon PI staining they will look like a bright punctate dot.

If we redefine the criteria we can have one more quadrant which is of AnxV +ve/PI +ve. Depending on temporal state the cell is at this can quadrant can be either apoptotic or necrotic.

Now I am saying all this as I have done live cell imaging with AnxV/PI and compared these different stages along with morphological changes (blebbing and swelling) in cells. If you are using adherent cells remember that you are now either scraping them or trypsinizing them before you read them on flow. This can stress your cells and push them towards death. In contrast, AnxV/PI is more advantageous for cells that are in suspension for example T cells.

Hope this helps.

Good luck,

Gaurav.

Anne von Koschembahr...Thanks for recommending that paper. This paper will answer most of your queries on partitioning an apoptotic cell from a necrotic cell and their overlap signatures through a flow cytometry experiment...I highly recommend it too...

I have some points that vary from what the people above said:

1) Only single AnxV+ cells can be considered to be apoptotic

2) AnxV+ / PI+ are necrotic (as defined by plasma membrane rupture). Even when cells die a necroric cell death and apoptosis is not involved, AnxV will pass the leaky plasma membrane and bind PS from the inside.

3) There is no late stage apoptosis. What people see is actually secondary necrosis. If you leave an apoptotic cell long enough in vitro, the plasma membrane will eventually rupture. This is not happening in vivo in health, since other phagocytes will clean up, before membrane rupture occurs. The prevention of a spill-over of intracellular material into the extracellular space is the whole prupose of the apoptosis program. If it is malfunctioning, it will lead to severe disease, like autoimmunity.

4) AnxV- / PI+ events are probably artifacts. If the membrane is leaky and PI can pass, AnxV should also be able to pass and stain PS from the inside, resulting in double positive events. Even the above cited paper "Discrimination between primary necrosis and apoptosis by necrostatin-1 in Annexin V-positive/propidium iodide-negative cells." says that this population propably occured due to the staining procedure. There could be a possibility that at a very early necrotic stage in a short time window the holes in the plasma membrane are so tiny that only PI can pass, but not AnxV. But this is an asumption.

5) (simplified)

double negative cells = healthy (non-apoptotic, no plasma mebrane rupture)

AnxV+ = apoptotic

double positive = necrotic (plasma membrane rupture)

AnxV- / PI+ = artifact (or see above)