Both possibilities could be true. In order to distinguish them you should run a time course: 1. If you see an early increase in Anexin+/PI- followed by an increase in Anexin+/PI+ you might have apoptosis. 2. In contrast, if you see a direct increase in Anexin+/PI+ without Anexin+/PI- you might have necrosis. However, you will need to confirm using inhibitors of caspases or necrostatin. Good luck!

As I understand you stain your cell with Annexin V and propidium iodide. Early apoptotic cells are stained by Annexin V but not PI because plasmic membrane is intact while externalizing AnnexinV. Late apoptotic and necrotic cells are stained by both PI and Annexin V. If you want to distinguish late apoptotic cells from necrotic ones you have to stain them with a Tunnel assay that stained DNA fragmentation a late apoptotic event.

Difficult to say with these stains. Ruptured cells will stain as double positive as the PI can enter the cell and AnnexinV can stain even exposed phosphatidylserine on the inner leaflet of the plasma membrane. It would be more definative to use a stain for active caspases to gauge of whether apoptosis has been triggered. One such option would be CaspACE (FITC-VAD-FMK) from Promega which works in flow cytometry or by fluorescent microscopy.

Both possibilities could be true. In order to distinguish them you should run a time course: 1. If you see an early increase in Anexin+/PI- followed by an increase in Anexin+/PI+ you might have apoptosis. 2. In contrast, if you see a direct increase in Anexin+/PI+ without Anexin+/PI- you might have necrosis. However, you will need to confirm using inhibitors of caspases or necrostatin. Good luck!

Hi,

Annexin V staining give better information about early/late apoptotic and necrotic cells death. you will try millipore MUSE Annexin V & Dead Cell Kit (Cat.# MCH100105). I have used this and got better result. I hope this will be usefull for you also.

All the best!!

If the cells are only PI+ then they are classified as necrotic cells but if the cells are PI+ as well as Annexin V +, the cells are classified as late apoptotic cells.

I agree with Sereno,

Double stained cells could be necrotic or late apoptotic (or there could be a mix of both). Early apoptotic cells have PS flipped to the outer leaflet where it can be bound by Annexin-V (PI is unable to penetrate the intact membrane so these cells will be Annexin-positive PI-negative). Once the membrane is permeabilised in necrotic/ late apoptotic cells Annexin-V can enter the cell and bind to PS on the inner leaflet as well as that on the outer leaflet, so you can no longer distinguish between the two types of cell death in this assay.

The above suggestions are very good; it is difficult to differentiate between the two populations ,i.e. late apoptotic and necrotic. A double stain (A+/PI+) is generally classed as late apoptosis and PI+ population is considered to be necrotic, but that is not essentially true. There are people who refer to a double stain as late apotosis/necrosis! You will have to perform a more detailed analysis such as time course and use specific inhibitors as suggested by Rodrigo. This should be relatively easier to do. For confirmation, you can always perform Tunnel assays. All the best.

If all you are seeing are double positive cells, there really is no way to distinguish between necrotic vs. late apoptotic cells. You will need to do a time course study where you would have annexin+ cells and PI- cells. Once you have those cells with few double positive it would be easier to make an argument that cells that are double positive are coming from the annexin+PI- population. If you do not see any accumulation of cells that are annexin+PI- then it is hard to claim any apoptosis. For technical reasons, I found that switching from PI to 7AAD is better for FACS because you would have an additional channel to use for staining (on most FACS machines PI uses FL2 and FL3 while 7AAD only uses FL3)

Ann/PI double positive cells can be both late apoptotic and necrotic cells. If you want to know the mode of cell death it is clearly detectable; with a good compensate strategy you never observe single Ann/+ cells during necrosis.

PI is membrane impermeant and excluded from viable cells, it does not pass through intact cell membranes.

Annexin-V is used as a probe to detect cells that have expressed phosphatidylserine on the cell surface, an event found in apoptosis as well as other forms of cell death.

Therefore, Annex-V-/IP-, Annex-V+/IP-, Annex-V+/IP+ and Annex-V-/IP+ populations corresponding to viable, early apoptotic, late apoptotic and necrotic cells, respectively.

Actually, in accordance with previous answer, you cannot say ANYTHING about the cells when they have passed to the Annexin V+/PI+ quadrant! The cells can have been dying due to necrosis (or other non-apoptoic ways of dying), apoptosis or a mixture of apoptosis/necrosis. You have to, as suggested previously, perform a very careful time-course to "catch" the window where you actually can see cells in the Annexin V+/PI- quadrant. Furthermore, since most kits provide you with Annexin V conjugated with fluorescein together with PI, you sometimes have considerable problems with spectral overlap between these two fluorochromes that is virtually impossible to compensate for. Therefore, it is better to use Annexin V conjugated with a fluorochrome that is excited by another laser (e.g. UV) than what is used for exciting fluorescein. There are some conjugated called something like Annexin V-647 or similar. You will probably find them... Good luck!

You are alos asking I suppose, if Annexin-V staining is related to late apoptosis, yes, it is... And I would say very late... as soon as PS are externalized the cells have almost undergo all the apoptotic signalling and are far beyond the point of no-return.

For the rest

AnnexinV-FITC+/PI- cells are apoptotic,

AnnexinV-FITC+/PI++ cells are primary necrotic (as staining control you can use cells that haven been treated By heat at 60°C for 30 minutes) and AnnexinV-FITC+/PI+ cells are secondary necrotic cells (or late necrosis)...

But if you have problems with that buy YO-PRO-1 and PI and double stain your cells to study their differential permeability... So YO-PRO-1minus/ PI- cel sare viable... and they pass to YO-PRO-1 + slowly and started t be slightly positives to PI- (apoptosis) and later oj you will get YO-PRO-1 ++ and PI ++ cells (necrotic after apoptosis and at the end the cells will lost their fluorecsence since they are totally permeable and non-stainable. Compare you results with annexin-V and calibrate.

This is far less expensive... (Molecular probes), don't buy the kits to do that (YO-PRO-1 1mg/ml) and PI (1 mg/ml for stock solution) used at 2 microg/ml for both.

Patrice

If you are interested in confirming a caspase-dependent cell death mechanism, you could test this pharmacologically using a caspase inhibitor. Treat your cells, prior to the addition of death activators with a caspase-inhibitor and follow your normal experiment protocol.

Cells undergoing caspase dependent death, would be expected to survive in the presence of the inhibitor. This may help you to identify apoptotic cells, however, there are other modes of death that could explain caspase-independent death such as necrosis or autophagy.

Goodluck

Hi Amir

You've asked what seems a sensible and simple question, however unfortunately the answer you settle upon will decide on how deeply you want to delve into this question.

Simplistic answer - Annexin V+/PI+ = both late apoptotic and necrotic.

Not so simplistic answer..

Annexin V/PI staining massively oversimplifies cell death, both programmed and not. It is not simply as clear cut as the cartoons will have you believe: apoptosis can occur without DNA laddering (in fact this is often the norm), by-and-large the upper echelons of the caspase cascade are more of a grey area than a certainty, autophagic cell death is not accounted for, and lets not talk about the secondary necrosis that can occur after apoptotic cells fail to be phagocytosed. I've listed several things that may be worth considering below..

Caveat 1:

Annexin V binds exposed PS as we all know, however it's worth remembering that any situation that allows Annexin V into the cell will also stain these cells (even, god forbid, 'early necrosis'). Also, whether PS flips in early apoptosis is strongly cell type dependent (ie, it's not necessarily the case in neurons).

Caveat 2:

PI staining is seen when both the outer and nucelar membranes have been compromised. Apoptosis by definition obstructs this from occurring (if you believe the purists), however I have a hard time believing that all apoptosis works perfectly and protects the DNA from PI. Likewise, I find it difficult to believe that a cell who's outer and inner membrane are compromised can survive as a cell (or cell remnant) for any substantial length of time (meaning if you really are detecting necrotic cells, you'll be detecting them in the most volatile phase of their short deaths).

Caveat 3:

Necrosis is generally a bucket term for instances where no evidence can be seen for an alternate cell death mechanism (namely apoptosis or autophagic cell death). Make sure you keep this in mind.

Hopefully there's something here of use. Good luck with your studies.

Al

Based on my experience I believe that the cells positive to both PI and Annexin-V are in late apoptosis. Moreover, this belief is supported by the publication of Troiano L. et al., Nature Protocols, 2: 2719-2727; 2007. To distinguish the two cell populations, I agree with Sereno Denis, and I suggest you to carried out a Tunnel assay. I hope to check out was helpful. Good job.

Thanks a lot dear colleagues for your perfect responses,

According to your explanations these cells could be late apoptotic or Necrotic cells and I should do more assay to find it out. But I still have a theoretical problem. If the essential goal of apoptosis incidence is to remove cells without any inflammatory response, how can we expect to see membrane permeabilization in late apoptosis that allows PI to staining the DNA? Can we see this step in vivo or just it happens in vitro when phagocytic system not exist? May be secondry necrosis is a better name for these cells?

This is really only something that you see in-vitro, by the late stages of apoptosis in-vivo, as the cell signals to other surrunding cells that it is undergoing apoptosis early on in the death process, and therefore these surrounding cells have already prepared to engulf that apoptotic cell's contents. For the necrotic cells, the immune system and related cells will have already begun to take care of the cell and phagocytize it before it breaks up into the cell debris like state you end up with at the very final stages in-vitro. One of the best ways to look at late stage apoptosis in-vivo, or really ex-vivo, is by taking tissue specimens, properly processing them and mounting them on slides for TUNEL assay, as well as many other anti-body targeted fluorescently tagged assays. One of the oldest and most reliable, as well as relatively easy to carry our staining protocols for invetigating tissue sections is H&E staining, but it does take a bit of practice to understand and identifiying the histopatholgy you are observing and often times it helps to have the assitance of a trained histopathologist to confirm your results. If you are dead set (no pun intended) on using a living animal model then there are several ways to go about this as well. Depending on the model you are using and the resources you have available to you, you should be able to design a method to target, stain and tag cells undergoing apoptosis and image using a Xenogen instrument for small animals. You could also use a doral skin fold window chamber model and image with a flourecence mciroscope using various fluorophores conjgated to anti-bodies of choice for looking at different proteins expressed in the late stages of apoptosis, as well as use other conjgated anti-bodies to look for the prescence of immune cells or cytokines in the case of in-vivo necrosis, which is something you will typically observe in the certian of a relatively large tumor mass.

Hi Amir,

I agree with Rodrigo Mora. First you should run a time course apoptosis induction and second check the stretch (concentration) of inductor. Do FACS at different time points. The cell type Is very important for an in vitro apoptosis analysis. I have experience in vitro apoptosis induction in T and myeloid cells. For thymocytes you can keep in culture short time and pLN T cells longer time. With aCD3/aCD28 and dexamethasone treatement you can see an early apoptosis increase (Anexin+/PI-) as soon as 2-3 h. You must have time 0 level of apoptosis. Over the time after 6h or O/N culture the balance change. You can see increase in percentage of late apoptotic/death (AnnexinV+/PI+) and less percentage of early apoptotic cells (AnnexinV+/PI-). If your experiment is working well you can see Annexin+/PI++ which can be dead/necrotic cells or PI+ annexinV – necrotic cells do to mechanical or physical damage. From my experience the best combination is 7AAD/annexinV, because 7AAD is more accurate than PI and helps to avoid non specific staining and as David Askew say, for technical reasons. I use often these combination that PI/annexinV. You can do 7AAD staining first, annexinV fitc second (in binding special buffer) and the best to do acquisition very fast (within 15min-45min). For PI staining you have to add maxim 10μl of working solution (50μg/ml) to the cells (max.1x10-6 in 250 μl Facs buffer) at the moment of acquisition, not before, vortex and acquire. Note: myeloid cells ex vivo or stimulated in vitro die very fast so you have to know the characteristics of your subset of cells.

I hope you get best results soon, good luck!

In case of your new question I think you should consider an alternative scenario. In fact, both viable and apoptotic cells have capability to uptake the DNA binding dyes but viable cells can engage membrane detoxification system to pump the dye outside the cells while the late apopotoic or necrotic cells with a problem membrane integrity can't.

There were some paper's in the 90's that used just 7AAD to look at apoptosis in lymphocytes (Was done to look at T cell apoptosis in AIDS patients). It used 20 ug/ml which is 20X higher than what is used for annexinV/7AAD staining. It took advantage of the change in membrane integrity of apoptotic cells vs. healthy cells vs. necrotic cells. You would compare FSC with FL3 and have three population of cells. the lowest FL3 would be viable, the middle population would be early apoptotic and the highest would be late apoptotic/necrotic population. The advantage of this protocol was that with the basic flow cytometers you could look at three other parameters (FL1,FL2, and FL4) because you were not needing to look at AnnexinV staining. Several paper by Hervé Lecoeur in this area.

I believe it's a late apoptosis cell not necrosis. necrosis population is PI+/AnnexinV-.

This is a question of cells in culture, since you induce apoptosis, and the cells are not digested by neighboring partners or macrophages in culture, you ended with a population A+/PI+, which are necrotic cells but indeed comes from the late apoptosis stage which is never attained in a tissue. You will find exactly the same type of situation if you use the very useful YO-PRO-1/PI staining... wit a YO-PRO-1 +/PI + population which is late apoptosis or necrosis.

annexin V is an initial step of apoptosis, even reversible sometimes. for late apoptosis you should look at caspase 3-8-9 or use TUNEL technique to detect DNA fragmentation, that is the lates stage...Tunel kit from Roche are the best.

I do believe that PI+/AnnxV+ events can be considered LATE APOPTOTIC cells, since they have postivie staining for PI (that means that their membranes are so damaged to let PI entering and binding DNA) but ALSO positive staining of ANNX V (that means that they have the typical features of apoptotic cells at hte membranes level as well).

PI+ events -in my experience- are usually considered "Necrotic" cells...theorically they are "fragments" of DNA bound by PI but without membranes..I agree with the other collegues that if you want to be sure of that you should perform a time-course TUNEL assay.

Actually, there is also a "physiological" (non in vitro-dependent) type of death that "produces" in this kind of analysis PI+ necrotic cells, and this is the "Mitotic Catastrophe": this kind of process occurs when cell is pushed to duplicate itself but it lacks the molecular tools to complete mitosis. In that case you will observe an increase in PI+ events rather than AnnxV+/PI+ events.

for late apoptosis you should use caspase 3 and/or TUNEL assay, otherwise you should get trouble publishing your data. be careful with compensation with PI too...

Annexin-V +/PI + cells can represent both late apoptotic and necrotic cells. You can use TUNEL assay to confirm late apoptosis(it is very reliable and easy). Maybe you can try performing the Annexin-V-PI assay at an earlier endpoint so you can detect early signs of apoptosis! Besides, compensation is a real issue in Annexin-V-PI assay, make sure you take it into consideration because personally I had some problems with this assay.

I couple week a go I do same assay and my Flow Cytometer do amazing work in separate all gate for normal, apoptotic, necrotics and dead cells,so My opinion is do to assay Anexin/pi and asalisis in citometer should be profesional work to separate all them cells.

PI+ (or 7AAD+) Annexin V + cells are dead cells (both necrotic and final apoptotic). If you look only to cell gate, they are necrotic. If you include also sub-cell population (lower FSC) they include apoptotic bodies as well. Generally, late apoptotic cells are AnnV+ PI intermediate to high.

btw, there is a kit from ENZO life sciences (check in your country) distinguishes necrotic cells with a specific dye for necrosis, ANNV and PI all in one. easy (if you have a FACS suitable for those colors).....

Attention to the terminology please! Late apoptosis with both annexin and PI positivity should be called secondary necrosis if the nucleus morphologhy suggests apoptosis (pyknotic nucleus, fragmented nucleus, condensed (brighter) chromatin etc). In the case of primary necrosis, there is nothing to do with apoptosis and the nucleus is expected to be enlarged and weakly stained (just opposite to being brighter) with nuclear stains due to excessive damage to/leakage of DNA. Of course, to distinguish these two events by performing what i have said above, you need a fluorescence microscope and to stain the cells with Hoechst dye while performing your staining with annexin V-FITC + PI. We routinely do this triple staining and always happy with the results. This means we quite reliably distinguish primary necrosis, secondary necrosis (late apoptosis), early apoptosis and control cells. We would be happy to email the staining protocol if you need. [email protected]

The cells are, by definition, necrotic. However, the key question is whether they have entered necrosis (i.e. have acquired a leaky plasma membrane) following apoptosis or not. If the necrosis has followed apoptosis then they are "secondary necrotic cell" where the necrosis is secondary to apoptosis. A view of nuclear morphology with DAPI can be a quick method for viewing to see if the cells are morphologically apoptotic. If the necrosis has not followed apoptosis then they are "primary necrotic cells".

Double staining (positive PI and annexin) is suggestive of secondary necrosis, single staining for annexin is suggestive of early apoptosis and single staining for PI is suggestive of primary necrosis. This is the simplest reliable approach to make an attempt of differentiation.

Dear Friends!

Please see the attached article which in a very nice way deals with the question how to intrerpret the Annexin V/PI-assay!

Thank you Dr Ulukaya for a very claryfying answer! I would like to ask you, however, is there a possibility to distingush between primary and secondary necrosis by using flow cytometery only? Or, is there really a true need for fluorescence microscopy?

Dear Michel Perego (and others)!

What you said about the kit with a "specific dye for necrosis" startled me a bit! Isn't it rather that the dye you refer to stains the whole nucleus (like e.g. PI), and that you then, as written by Dr Ulukaya, still have to judge the nuclear morphology by fluorescence micropscopy for distinguishing between primary necrosis (true necrosis) and secondary necrosis (resulting from preceeding apoptosis), or..!?

Its quiet an active discussion. My opinion is that if you really need to distinguish between apoptoic and necrotic (true necrotic) cells, you should do either a morphological analysis under a microscope or some specific assays for necrosis. Its practically not possible to distinguish apoptotic and necrotic cells with Annexin v-FITC-PI staining. But it can easily points out cells before entering apoptosis or necroses ( Annexin V positve, PI negetive).

Dear Thomas,

Terribly sorry for too late answer as i really have noticed it right now. It is possible to distinguish primary necrotic and secondary necrotic (also called late apoptotic) cells by flow cytometry. When annexin V-PI is used, left upper quadrant belongs to the primary necrotic cells (area of higher PI and lower annexin V level), while the right upper quadrant (area of higher PI and higher annexin V level) belongs to secondary necrotic cells. So, if the quadrants are clearly differentiated from each other, then maybe no need to use flourescence microscopy additionally?. All the best...

It is typically accepted that there are four populations from this assay. Q1 (Annexin V negative, PI positive) - necrotic cells; Q2 (Annexin V positive, PI positive) - middle to late apoptosis; Q3 (Annexin V negative, PI negative) - live cells; Q4 (Annexin V positive, PI negative) - early apoptosis.

Without looking at the cells under the microscope to observe their morphology, there is no way to 100% verify via FACS. The exception is if you or your institution has an ImageStream instrument, where it does FACS and microscopy at the same time.

Dear Engin Ulukaya：

Is there any references to support your view on that left upper quadrant belongs to the primary necrotic cells (area of higher PI and lower annexin V level), while the right upper quadrant (area of higher PI and higher annexin V level) belongs to secondary necrotic cells.

Both Annexin-V and PI positivity in the population include the possibility of late apoptotic and necrotic population. As apoptotic and necrotic cells slightly differ in their morphology, a smart way to distinguish between them is with the help of FACS data analysis software like FloJo, gate the both positive population and then draw an SSC vs HSC. You will be able to see two different populations in the plot.

FYI-The appearance of phophatidylserine (PS) on the outer leaflet of the plasma membrane and the loss of membrane phopholipid asymetry occurs rather early during the sequence of apoptotic events and results from amino-phospholipid translocase downregulation and scramblase activity. This can be detected by the binding of annexin V to PS, which also requiresthe presence of calcium ions. The "flipping" precedes DNA fragmentation for example, and is untypical to necrosis.

Thanks dear Sharona Even-Ram :

Although the events you mentioned are typical for apoptosis, in a necrotic cell there is an energy crisis which can disturb enzymatic activity such as translocase, is not it true? On the other hand, there is a technical problem that in apoptotic cells, plasma membrane is remained intact while in necrotic cells the membrane integrity disturb which can provide enough space for Annexin V entrance and binding to internal PSs. Is this not possible?

If we accept these facts, I think this technique can not help to distinguish apoptosis from necrosis in this way.

in necrotic cells, if annexin V can reach the inner leaflet, PI will do so as well, so that's why double positive ones should excluded. Translocase downregulation is part of the active process and not due to energy crisis. The fact that cells maintain membrane integrity is the advantage, not the problem...

The problem is that if you look late into the process, dead cells areafter all dead cells and you will not be able to tell the cause of death by this technique

I mean, Translocase inactivation for energy crisis in necrotic cells is equal to Translocase downregulation in apoptotic cells and both of them can lead to PS appearance in outer leaflet. Also, loss of membrane integrity in necrotic cells can make a problem because these cells can obtain annexin V binding to inner leaflet PSs, that is seem similar to apoptotic cells and this phenomenon can disturb the possibility of recognizing these two types of cell death. But, I totally agree with you at the end because this technique is not applicable for distinguishing the reason of death in cells unless using fluorescent microscopy to see fragmented nucleus instead of general flowcytometery analysis.

I think the cell morphology is very important in detecting apoptotic cells, charachterised by cell shrinkage, nuclear condensation, DNA fragmentation and formation of apoptotic bodies at the end. AnnexinV/PI flowcytometry is good for detecting early apoptosis and when studying the kinetics of apoptosis as you mention early (early membrane changes of PS flipping to the exterior of the cell membrane) and late stages. And as many have pointed in this very nice discussion, late apoptisis and secondary necrosis may overlap, as found in the upper right quadrant in the graph in flowcytometry. When there is membrane permeability and PI staining necrosis has likely ensued. Thus, it is always better to use additional assays, particularly, classical light microscopy, Flourescent microscopy, extensive FCM for studying nuclear changes, and DNA fragmentation confirming apoptosis. The refs. below is an article from 1995, but describes the methods well. May help in understanding the detection of apoptosis versus necrosis.

http://doc.utwente.nl/74240/1/JIM1995.pdf

Hi Amir,

Distinguishing apoptotic from necrotic cells is not trivial, as there is no positive marker for necrotic cells so far. The only way to really identify the celll death type is by combining several methods: (electron) microscopy, enzymatic assays/Western blot and inhibitors where possible. The features change over time so it it is important to make measurements at different timepoints in the cell death process.

We just published a book chapter on how to discriminate between different cell death types (see fow chart atached).

I can send you a repreint if you'rre interested.

best regards,

sasker

Forgot the flow chart... Here it is

Dear Sasker!

As I previously have been a participant of this very important discussion, I would very much appreciate if you had the opportunity to send me a reprint of the book chapter you mention. My mail is: [email protected]

kind regards

/Thomas

Hi amir

i measured apoptotic cells by AnnexinV/PI by flowcytometry. Annexin V+/PI- are early apoptotic and AnnexinV+/PI+ are late apoptotic or necrotic cells.

Dear Sasker!

I would appreciate if you had the opportunity to send me a reprint of the book chapter you mention. My mail is: [email protected]

kind regards

Anita

Hi Sasker

I would appreciate if you can send me a reprint of the book chapter. My mail is: [email protected]

Thanks

Dear Sasker

I would appreciate if you could send me a reprint of the mentioned book chapter as well. My mail is [email protected]

Thanks

Best, Jorrit

Dear Sasker

I would appreciate if you could send me a reprint of the mentioned book chapter as well. My mail is [email protected].

Thanks,

kind regards

Dear Sasker

I would appreciate if you could send me a reprint of the mentioned book chapter as well. My email address is [email protected]

Thanks

Regards,Neeta

Dear Sasker

I am analyzing the cell death for early or late apoptotic cells. I would appreciate if you had the opportunity to send me a reprint of the book chapter you mention. My mail is: [email protected]

thanks a lot.

Tong 

Late apoptosis mean Annexing V positive , PI negative whereas necrosis mean only PI positive . May be use TEM electron micrograph confirm your data study internal structure 

Dear Sasker I would appreciate if you could send me a reprint of the mentioned book chapter as well. My mail is [email protected] Thanks Best, Haili

Dear members

annexin V+/PI+(late apoptosis) and necrotic(annexin V-/PI+) cells was calculated with FACSDiva software,version 6.1.1(BD,Biosciences) from Heliyon 4 (2018) e00638. doi:10.1016/j.heliyon.2018. eoo638