How to differentiate apoptotic from necroptotic cell death? Part III.

How to determine cell death?

Various assays exist to determine cell death and all, unfortunately, have their limitations. In this section, I’ll discuss all of the assays that I’ve got experience with. I’ll also indicate whether I deem an assay suitable for high throughput screening (HTS) or not. In general, I’d advise against using an assay that you don’t understand the principles of. Companies such as Promega are never very eager to reveal the underlying principles of their assays, as they’re afraid other companies will copy them, but if you don’t know what, for example, the ‘live-cell protease’ activity is that an assay such as Promega’s  MultiToxFluor assay measures, you can’t possibly determine whether your treatment is indeed affecting the cells’ health or just the activity of this mystery protease. If a company won’t tell me what it is exactly that their assay does and what the buffer components are, I won’t trust that assay.

MTT assay

Figure 1. MTT assay to determine TNFa toxicity on L929 cells.

A rather old fashioned method for determining cell death is the good old MTT assay. This assay depends on the reduction of tetrazole to formazan by oxireductase enzymes in living cells. Formazan forms a purple precipitate that can easily be detected with an absorption spectrometer. However, just like ATP assays, this assay also detects loss of cells, lack of proliferation or reduced metabolic activity, rather than cell death itself. MTT assays are certainly useful because they’re extremely cheap and easy to perform, but shouldn't be used to accurately determine cell death, let alone differentiate between modes of cell death. In addition, the assay only really works well for adherent cells and the cells are lysed in the process and the assay can therefore not be used in a multiplex set up.

Detection method: Absorption

Pros: Cheap and relatively easy

Cons: Sensitivity, not a cell death assay, doesn't discriminate, samples destroyed in the process

Crystal Violet

What I like about using crystal violet is that the assay doesn't depend on metabolic activity of the cells, but exclusively on the number of adherent cells remaining in your well. In addition, the morphology of the stained cells can easily be judged by simple light microscopy. Since the dye can be re-dissolved with methanol after staining and washing, the assay can also be used to accurately quantify the number of cells remaining in your well (Figure 2). Drawbacks are, of course, that the assay can only be used for adherent cells, that the cells are fixed during staining and that it’s hard to integrate the assay in a multiplex procedure. Since the assay only determines loss of cells (which can also be lack of proliferation) no conclusions about the mode of cell death can be drawn from crystal violet staining alone. I find this assay particularly useful for illustrating colony outgrowth after treatment, though it can also be used for routine screening. Because the cell mono-layer easily gets damaged in the execution of the assay during washing, fixing or staining, the assay is not very suitable for small well formats. It works well enough in larger wells, down to a 24-well format, since the relative contribution of some minor damage to the mono-layer won’t influence the outcome of the assay as much in larger wells as in smaller wells.

Figure 2. Crystal violet assay. A: Titration of L929 cells stained with crystal violet to generate a standard curve on a double log scale. Insert shows a macroscopic view of the wells with increasing amounts of cells. B: The reactive oxygen species (ROS) scavenger butylated hydroxyanisole (BHA) protects L929 cells from TNF/zVAD-induced necroptosis. However, higher concentrations of BHA are toxic. C: Macroscopic (top) and microscopic images (bottom) of L929 cells untreated (left), treated with TNF/zVAD (middle) or TNF/zVAD+necrostatin-1 (right). Cells were treated for 4h after which the medium was refreshed and cells were allowed to grow for a week.

Detection methods: Light microscopy, absorption (after re-dissolving dye in methanol)

Pros: Very cheap and relatively easy, objectivity

Cons: Sensitivity, not a cell death assay, doesn't discriminate, only works for adherent cells, cells fixed in the process, prone to errors when the cell mono-layer is scratched, toxicity of the reagents

ATP assays

Luciferase-based assay that determine cellular ATP levels, such as Promega’s Cell Titer Glo, have become very popular, especially in high throughput screens. Ease-of-use is the main selling point of these assays, as they only require a single reagent addition and a short incubation time. In addition, the assay is very sensitive. However, this assay does not determine cell death. Rather, a reduction in ATP levels can reflect several scenarios. First, a reduction in ATP levels in a given well might indicate a reduction in cell numbers as a consequence of death, but could also reflect a reduction in cell growth. If the treated cells expanded more slowly than the control cells, this would be reflected in a relative reduction in ATP levels. Second, overall ATP levels may be transiently reduced in cells under stress without this resulting in cell death. Third, during apoptosis ATP levels actually increase before cell death occurs. Thus, cell death assays based on determining relative ATP levels can very easily lead to invalid results and wrong conclusions. For an initial screen, such an assay might be useful but one should be extremely cautious to draw conclusions based on results obtained from ATP assays alone.

Detection method: Luminescence

Pros: Easy, single addition, suitable for HTS, very sensitive

Cons: Not a cell death assay, doesn't discriminate, samples destroyed in the process

Dye exclusion

In many older papers ‘apoptosis’ is equated with cell permeability for DNA-binding dyes such as propidium iodide (PI) or ethidium bromide (EthBr). However, apoptotic cells only become permeable to such dyes at a very late stage, while necroptotic cells lose plasma membrane integrity much earlier. Thus, dye uptake is either a sign of necroptosis, primary necrosis or late-stage apoptosis (Figure 3). Bear this in mind when reading older papers and don’t make this mistake yourself. Of course, this is not only true for old-fashioned dyes such as PI, but also for newer dyes such as the Sytoxdyes from Life Technologies (formerly Molecular Probes/Invitrogen). Although dye exclusion by itself has limited usability, it’s a method that can easily be combined with other methods in a multiplex assay, as I’ll discuss below.

Detection method: Fluorescence

Pros: Cheap and very easy, single addition and no washing required, suitable for both adherent and suspension cells, many dyes available in different colours, suitable for HTS

Cons: Only indicates necrosis or necroptosis, not early-stage apoptosis

LDH Release

Figure 3. Sytox green staining vs. LDH release from U937 cells
treated with TNFa in the presence of increasing amounts
of zVADfmk for 24 hours.

When cells lose membrane integrity, their content is spilled in the environment. This includes the enzyme lactate dehydrogenase (LDH). Activity of this enzyme can easily be detected with a commercially available colorimetric assay. The great advantage of this assay is that only the culture medium of the cells is required and that the cells themselves can therefore be used for other assays, for example Western blot or FACS. I found this assay to be very similar in sensitivity to dye exclusion (Figure 3). Of course, just like dye exclusion, the assay only determines the rate of necrosis which can be either a consequence of necroptosis, primary necrosis or secondary necrosis in late-stage apoptosis. An advantage over dye uptake is that you only need an absorption reader for detection and these are usually cheaper than fluorescence readers, although the assay itself is somewhat more expensive. 

Detection method: Absorption

Pros: Cheap and very easy, single addition to culture supernatant, cells can still be used for continued culture or other assays, suitable for both adherent and suspension cells

Cons: Only indicates necrosis or necroptosis, not early-stage apoptosis

Resorufin/Resazurin

The principle of this assay, sold as AlamarBlue or Cell Titer Blue, is the conversion of blue, non-fluorescent, resazurin to red, fluorescent, resorufin by living cells in an oxidation reaction. The assay has several advantages: the cells are not destroyed in the process, the results can be measured with either a fluorometer (red) or an absorbance spectrometer (red/blue) and can easily be combined with other assays that utilize different fluorescence wave lengths or luminescence. In addition, the results are aesthetically pleasing (Figure 3).

Figure 4. Cell Titer Blue on U937 cells treated with various stimuli/inhibitor titrations in a 96-well plate (A). Wells with living cells turn pink, others remain blue. In B I plotted the rate of resorufin turnover by cells treated overnight with TNFa in the presence of increasing amounts of zVAD versus Sytox Green staining.

However, just like ATP assays and such, the assay doesn't indicate cell death but rather cell metabolism. Since different cells display different rates of oxidative metabolism under different circumstances, it will take some experimenting before reliable results can be obtained from this assay. I found that it takes jurkat or U937 cells about 2-4 hours to metabolize resazurin, but I found that L929 cells act rather unpredictably in this assay. The assay can be used as either an end-point read out or kinetic read out. In the form of Amplex Red, resorufin can also be used to determine a rapid oxidative burst. Back when I worked on neutrophils we used Amplex Red routinely to determine neutrophil responses to certain stimuli.

Detection method: Absorption or fluorescence

Pros: Fairly cheap (I only once got a 10 mL sample of Cell Titer Blue and used it for years) and easy, single addition followed by incubation and measurement, suitable for multiplex assays, suitable for HTS, cells remain alive and intact

Cons: Indicates oxidative metabolism, not a cell death assay, doesn’t discriminate

Annexin V Binding

Probably my favourite apoptosis assay is binding of fluorescently labelled Annexin V binding followed by flow cytometry (FACS). Annexin V binds to phosphatidylserine (PS) which is exposed on the outer membrane of apoptotic cells. PS exposure is a passive process and happens when a cell’s active mechanism for retaining PS on the inside of the plasma membrane (the enzyme family collectively known as ‘flippases’) is compromised. Flippases are ATP-dependent enzymes that are sensitive to Ca²⁺. Thus, a drop in cellular ATP levels or a rise in intracellular Ca²⁺ levels will inhibit flippases and lead to PS exposure.

Figure 5. Membrane asymmetry in healthy cells versus PS exposure in apoptotic cells. An image I drew years ago.

Not too much is actually known about the regulation of these flippases during apoptosis, but PS exposure certainly is a very accurate hallmark of early apoptosis. However, PS exposure isreversible, some cells (such as macrophages) constitutively bind low levels of Annexin V and PS exposure can occur under certain rare  conditions (such as Barth syndrome) in the absence of apoptosis. Nevertheless, I’ve generally found Annexin V staining to correlate nicely with apoptosis. Of course, when the cell membrane integrity is compromised, Annexin V will also enter the cell and stain both sides of the membrane. Thus, high Annexin V staining alone can be an indication of either apoptosis or necrosis. Whatever the case, when a cell displays high Annexin V positivity something’s wrong. Annexin V binding can (and should) easily be combined with dye exclusion for accurate differentiation of (early) apoptotic cells and necrotic cells. Bear in mind that Annexin V binding is Ca²⁺-dependent and your binding buffer should therefore always contain ~2.5 mM CaCl₂. If you wash away the Ca²⁺, the Annexin V will also fall off. Finally, living cells will constantly expose low amounts of PS that are actively transported back in side and therefore constant exposure of living cells to Annexin V will very slowly lead to the uptake of the Annexin V and the staining of the cells. However, if you keep the cells on ice, you effectively fix the plasma membranes and the PS levels in the outer membrane won’t change anymore, even if you leave the cells unfixed.

Detection method: Fluorescence

Pros: Accurate assay for apoptosis, sensitive, easily combined with other assays

Cons: Indicates both early apoptosis and necrosis, not suitable for HTS

Figure 5. Annexin V staining of jurkat cells either deficient in FADD (DEF) or reconstituted with FADD (REC) treated with TNFa or TRAIL in the presence of the indicated inhibitors. Samples were taken every 2 hours, stained with Annexin-V-FITC and analyzed by flow cytometry.

Caspase activity

Caspase activity can be determined in a variety of ways and is a fairly reliably indicator of apoptosis. Of course, as I mentioned in the first post of this series, caspases are not exclusively activated during apoptosis and it’s not trivial to tell the activity of one caspase accurately apart from the activity of another caspase. However, caspase-3 activity in particular is certainly a hallmark of apoptosis. Thus, you’ll always find caspase-3 to be very active in apoptotic cells, although limited caspase-3 activation can occur in non-apoptotic cells. To determine caspase-3 activity any assay containing the tetra-peptide substrate ‘DEVD’ will do. I prefer Ac-DEVD-AFC over AMC labelled substrates, since they seem to be more sensitive and AFC will also turn yellow when released from the DEVD moiety, which can even be detected by absorption. Those are available as fluorescent or luminescent assays but you can also buy the substrate and make your own lysis buffer. The substrate is also somewhat cell permeable and can therefore be added to cells before inducing apoptosis and then be used to kinetically determine the increase in apoptosis in a well. However, such an approach is more likely to detect late-stage apoptosis, when the plasma membrane of the cells becomes compromised. In addition, the DEVD is also consumed by the proteasome, so healthy cells will hydrolyse it very slowly.  Therefore, rather than stating that you’re measuring caspase-3 activity when using DEVD as a substrate, state that you’re measuring DEVDase activity as you can’t be absolutely certain that the activity you measure is derived from caspase-3. Still, a fluorescently labelled tetra peptide substrate can easily be combined with dye exclusion and viability (resazurin/resorufin) to determine whether your cells have become apoptotic or necrotic after treatment.

If you want to obtain more accurate information about the particular caspase involved, you could consider using an antibody that only detects the cleaved for of the caspase, tag it with a fluorescent label and perform flow cytometry. However, only the executioner caspases (3, 6 and 7) require cleavage for activation and the available antibodies detect other proteins with the same cleavage site as well. These are often cleaved as a consequence of caspase activity (caspases like to cleave their own linkers and will cleave every other protein with the same epitope as well) which is why Western blots with active caspase antibodies will often show a large amount of bands. In flow cytometry or microscopy assays you get no information about the size of the proteins labelled and therefore no accurate information about whether you’re really looking at the caspase or a product of caspase activity. A better method to determine which caspase has been activated is to label all active caspases with a biotinylated substrate, such as bVAD, bEVD or bVEID, perform a pull-down with streptavidin beads and detect your active caspases on Western blot.

Figure 6. Active caspase detection in cell extracts. Extracts were activated by addition of cytochomre c and caspase activity was detected by hydrolysis of the substrate Ac-DEVD-AFC (A) at the indicated time points or active caspases were labeled with bEVD-AOMK, pulled down with streptavidin beads and analyzed on Western blot (B). Active caspase-6 and -3 could be detected. Caspase-8 is cleaved (by caspase-6) but not activated under these conditions. See van Raam et al. for further details.

Detection method: Fluorescence, luminescence or absorbance

Pros: Accurate assay for apoptosis, sensitive, suitable for multiplex, suitable for HTS

Cons: Indicates mostly late apoptosis, risk of false positives

The ultimate multiplex assay?

In a good multiplex assay, you want to combine at least one parameter to detect necroptotic cells and one to detect apoptotic cells. I generally prefer to combine Annexin V binding with dye exclusion on a flow cytometer, as flow cytometry provides a very versatile platform and also provides you with valuable information about cell morphology, besides fluorescence. I've personally come to prefer FITC-labelled Annexin V (I get it from Bender Med, now eBiosciences) with Sytox Red. FITC fluorescence and Sytox Red are excited by different lasers and there’s therefore no need for compensation, while PI and FITC are excited by the same laser and their fluorescent peaks are close together. However, this assay is less suitable for HTS, although most steps can easily be automated. I know the Vandenabeele lab has developed an assay wherein they combine Sytox Green with Ac-DEVD-AMC to detect caspase activity. This seems to work well for them, although I’d combine it with a viability assay in the form of Cell Titer Blue. Assays that can be performed kinetically are always superior to end-point assays, but in the end the use of inhibitors can provide you with the most accurate information.