Apoptosis is the “classical” form of programmed cell death, which plays an important role in the maintenance of biological cells and systems. This article focuses on apoptosis, with an overview of necrosis and other forms of programmed cell death: anoikis, pyroptosis, parthanatos, and ferroptosis. Dedicated review articles exist for neutrophil extracellular traps, including both cell death of neutrophils and monocytes, and autophagy.

Apoptosis is characterized by the activation of initiator and effector caspases, cellular shrinkage, chromatin condensation, membrane blebbing, loss of mitochondrial integrity, and DNA fragmentation. It is a tightly regulated and controlled active process, ultimately leading to the breakdown of a cell while avoiding an inflammatory response. Once broken down, cellular fragments (apoptotic bodies) are taken up into phagosomes. Apoptosis can be segregated into extrinsic (the activation of cell surface receptors such as TNF alpha which leads to the activation of caspase-8 [1] ) and intrinsic pathways, which can be distinguished by activation of caspase-9. Many aspects of apoptosis are regulated at the mitochondria by several families of proteins, including SMACs (small mitochondrial-derived activator of caspases), IAPs (inhibitor of apoptosis proteins), and the Bcl-2 family, as well as membrane polarity and integrity. Table 1 lists some of the commonly studied proteins (among at least 400 genes) involved in the apoptosis process.

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Figure 1. Characteristic morphological changes of apoptosis

Apoptosis is tightly regulated and consists of several ordered, sequential steps. For example, the activation of an initiator caspase (caspase-8 or -9) comes before the activation of the effector caspases-3 and -7, which precedes DNA fragmentation. This means that the timing of any apoptotic assay is critical, and the testing of numerous time points is usually required to observe the appropriate changes in a meaningful way.

Apoptosis is characterized by nuclear and cytoplasmic condensation and fragmentation, membrane blebbing, and the formation of apoptotic bodies. Although not accurately quantifiable, these changes can be visualized microscopically at a basic level by light microscopy (Figure 1), and at a more fundamental level using electron microscopy or time-lapse microscopy.

The activation or cleavage of caspases is one of the most widely used methods for assessing apoptosis in cell cultures. Apoptotic caspases can be divided into two groups based on their structure and role in the cell death process. Specifically, initiator caspases possess a CARD (caspase recruitment domain) or DED (death effector domain; Figure 2) in their prodomain, while effector caspases only have short prodomains. Initiator caspases (caspase-2, -8, -9, and -10) are responsible for initiating specific caspase cascades. Because of this, these proteins can be used to distinguish between the different apoptotic pathways: caspase-8 is activated by extrinsic death receptors, while caspase-9 is involved in intrinsic mitochondrial apoptotic pathways. The activation of effector caspases (caspase-3, -6 and -7) by cleavage at specific Asp residues triggers events, ultimately leading to the breakdown of the cell. The process of caspase activation is outlined in Figure 3.

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Figure 2. The structure of caspases

There are now multiple methods for assessing the activation of caspases, ranging from fluorescent, colorimetric and luminescent substrates that are added to cells, to western blots and ELISAs for cleaved caspases that can be used on cell extracts and fixed cells, or tissues by immunocyto- or immunohistochemistry. For example, Genet G et al stained HUVEC cells with an anti-cleaved caspase-3 antibody to measure the apoptosis [2]. Assays involving caspase substrates are simple and usually in a microplate format. A combined cell lysis and activity buffer containing pro-fluorescent, pro-colorimetric, or pro-luminescent caspase substrates are added directly to the culture medium of cells in culture. Active caspases in the sample then cleave the substrate, resulting in a product that fluoresces or luminesces when excited at the appropriate wavelength. Examples of substrates for the key caspases discussed here are described in Table 2.

Several specific caspase inhibitors are available to use as controls to demonstrate the specificity of the respective substrates. Examples include Ac-DEVD-CHO (caspase-3/7 inhibitor), Ac-DEVD-FMK (pan-caspase inhibitor), Z-IETD-FMK (caspase-8 inhibitor), and Z-LEHD-FMK (caspase-9 inhibitor). Together these reagents form a powerful tool for assessing apoptosis, although consideration must be given to which substrate is more appropriate for your needs.

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Figure 3. Schematic representation of apoptotic pathways. Reproduced with permission from Favaloro et al. (2012) Aging 4:330-9

Aside from the obvious availability of equipment, other important considerations include sensitivity and the output of the substrate used. Colorimetric substrates form a colored product that absorbs light in the visible range. The signal obtained from the assay is directly proportional to the amount of analyte being assayed. These assays can be carried out in clear plates, and the substrates are generally more stable and more cost-effective than either fluorescent or luminescent substrates. A fluorescent substrate produces a reaction product that fluoresces when activated by a particular wavelength of light, and the RFU (relative fluorescent units) detected are proportional to the analyte being measured. These substrates are more prone to quenching, signal interference from assay conditions and physical aspects such as fingerprints, and are only slightly more sensitive than colorimetric assays. However, they significantly broaden the dynamic range of assays allowing very high readings, since they are not subject to the OD 2-4 limit forced on colorimetric assays. Fluorescent assays should be carried out in plates with black wells to prevent the fluorescent signal from leaching. Luminescent caspase substrates are based on a bioluminescence compound such as firefly luciferase. Luminescence occurs from the emission of light from the luciferase as it returns from an electronically excited to the ground state. These reactions are carried out in white-walled plates, and are typically several-fold more sensitive than fluorescent substrates, have a low background, and the methods involved can be faster. However, the substrates, like fluorescent substrates, can be very expensive.

In addition to assays to assess activity, several high-quality antibodies for the major caspases (-3/7, -8 and -9) are also available. These antibodies can detect full length (inactive), full length and fragments, or only fragments (active), allowing them to be selectively used in western blotting, immunocytochemistry, and immunohistochemistry. For instance, activated caspase 3 was used as a marker for apoptotic cells and neuronal loss [3, 4].

The breakdown of cellular chromosomes is a critical step in apoptosis, and there are numerous ways in which this can be quantified. Historically, DNA fragmentation was assessed by simple gel electrophoresis, and this is still used today in certain areas. Although this method informs you of DNA breakdown, it is not easily quantifiable. Instead, there are several ELISA kits available that measure the fragmentation of DNA. These kits involve detecting BrdU-labelled fragments released from damaged cells in a sandwich ELISA format, giving data that can be quantified and analyzed statistically. Similarly, kits can be used that specifically detect the fragmentation of histone-associated DNA in the cytoplasm of cells, using a histone-capture antibody to capture nucleosomes to the microplate, followed by a DNA secondary antibody.

Other methods to assess DNA fragmentation take advantage of enzymes capable of adding labeled nucleotides to the end of DNA fragments. The traditional method using this technology is TUNEL (terminal UTP nick end labeling), which uses terminal deoxynucleotidyl transferase (TdT) to add fluorescent or colorimetric labels to the recessed or blunt ends of DNA. TUNEL is routinely used to assess apoptosis on tissue sections, but can also be used with immunocytochemistry in cell cultures, flow cytometry or in situ fluorescence detection. TUNEL assay can also be used to assess chromatin accessibility after the treatment of DNase I [5].

The Bcl-2 family of proteins is an important regulator of mitochondrial-mediated apoptosis. The family comprises both pro- and anti-apoptotic proteins that work in concert to regulate the potential of the outer mitochondrial membrane (several comprehensive reviews on this subject have been written [6-8] ). Although Bcl-2-family proteins are important apoptotic regulators, measuring their absolute levels is not an indicator of apoptosis. Instead, their phosphorylation state and/or conformational changes are what are important. Key members and the changes that can be assessed are summarized in several excellent reviews [7, 9].

These changes can be measured by several methods, including basic immunoblotting, ELISA or conformational immunoprecipitation for proteins such as Bax. Assays take advantage of a conformational change that occurs when Bax gets activated, ready to insert into the mitochondrial membrane. This conformational change exposes the N-20 epitope, a 20 amino acid region at the N-terminus of the protein. If Bax is immunoprecipitated in a mild, CHAPS-based buffer, the conformation of the protein remains unchanged. This can be exploited to determine the ratio of total to active protein.

Apoptosis is also accompanied by alterations of mitochondrial membrane potential (Δψm), which can be caused by either the formation of pores by members of the Bcl-2 family or the opening of the inner membrane permeability pore complex (PTPC). This can be assessed using fluorescent probes in live cells. Examples of dyes include rhodamine 123, TMRE, and JC-1. These dyes are typically lipophilic and cationic, meaning that mitochondria with a more negative Δψm (polarized; healthy) will accumulate more dye than one that is depolarized in unhealthy mitochondria [10]. These fluorescent dyes have to be utilized and optimized carefully to ensure that the interpretation of the data is correct, and the selection of dye will depend on the question being asked. For example, JC-1 is commonly used to assess Δψm, leading to apoptosis. When accumulated in mitochondria, JC-1 exhibits a shift in fluorescence emission from green (~529 nm) to red (~590 nm). This allows mitochondrial depolarization to be measured by a decrease in the red/green fluorescence intensity ratio, which can be assessed by fluorescence microscopy or FACS. TRMR/TMRE and Rhod123 are commonly used for slow and fast resolving acute studies, respectively [11], and are usually monitored by flow cytometry using co-incubation with mitochondrial-specific probes such as rhodamine 123 or MitoTracker.

Cytochrome c is an essential part of the electron transport chain and is found associated with the inner mitochondrial membrane and in the intermembrane space in healthy cells. Upon the initiation of apoptosis, cytochrome c gets released into the cytoplasm, where it binds to Apaf-1 (apoptotic protease activating factor-1) leading to the cleavage and activation of caspase-3. As such, detection of cytochrome c release is a useful tool for assessing early-stage apoptosis. There are many different ways to measure this, and these methods vary in their sensitivity, accuracy, and the amount of time taken to assay. Firstly, cell cultures can be fractionated into cytoplasmic and mitochondrial compartments, and cytochrome c can be assessed by immunoblotting, and the same antibodies can be used for immunocytochemistry. Both of these techniques are labor-intensive, and care must be taken to use the appropriate controls when carrying out cell fractionation to ensure there is no contamination of cellular compartments. Cytochrome c release can also be detected using flow cytometry after selective permeabilization of the plasma membrane with digitonin [12].

Intracellular calcium plays a key regulatory role in numerous biological processes, including apoptosis [13]. A rapid rise in mitochondrial calcium levels is a pro-apoptotic signal that causes swelling of the mitochondria, leading to disruption of the outer mitochondrial membrane. This culminates in the release of cytochrome c and other apoptotic factors including AIF and Smac/DIABLO into the cytoplasm. Changes in mitochondrial calcium levels can be measured in live cells using synthetic dyes such as rhodamine-2 or rhodamine-FF. However, these techniques are challenging since the dyes can leak out of the mitochondria, can lose effectiveness over time, and can themselves disrupt the morphology of mitochondria [14]. The most accurate way to assess mitochondrial calcium levels is, therefore, to use fluorescent indicators specifically targeted to the mitochondria combined with fluorescence microscopy. There are several examples of such dyes, including radiometric pericam [15]. Although very useful for studies relevant to mitochondrial metabolism and signaling, using mitochondrial calcium levels as an indicator, specifically of apoptosis is not ideal due to the complexity of the experiments, and there are many more straightforward ways of directly assessing apoptosis.

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Figure 4. Annexin V/PI FACS Images. Image provided courtesy of Abcam Inc. Copyright©2013 Abcam

Several methods are available to measure alterations in plasma membrane integrity. Phosphatidylserine (PS) is a negatively charged membrane phospholipid normally found exclusively in the inner leaflet of the plasma membrane. During the early stages of apoptosis, the charge ratio of the membrane gets disrupted, and PS becomes exposed to the cell surface [16]. This can be detected by flow cytometry using Annexin-V, a phospholipid and calcium-binding protein [17], conjugated to FITC, or an alternative fluorochrome. These assays are usually combined with propidium iodide or 7-aminoactinomycin D (7-AAD) to assess membrane integrity concurrently. This allows the distinction between healthy (Annexin V and PI negative; Figure 4 lower left quadrant), early apoptotic (Annexin V positive, PI negative; Fig 4 upper left quadrant), necrotic (Annexin V negative, PI-positive; Fig 4 lower right quadrant), or dead (Annexin V and PI-positive; Fig 4 upper right quadrant) cells. For example, For example, Calvanese V et al combined 7-AAD and annexin-V-PE staining in flow cytometry to assess the apoptosis of cultured hematopoietic stem or progenitor cells [18]. Boettcher S et al assessed daunorubicin-induced apoptosis by flow cytometry using Annexin V APC staining from Biolegend ( 640920 in combination with DAPI from Biolegend ( 422801) [19].

As part of the apoptotic process, additional cellular proteins get broken down and can be used as apoptotic markers. One of the best examples of this is PARP cleavage. PARP (poly ADP ribose polymerase) is a family of at least seven nuclear proteins. Upon single-stranded DNA breaks, PARP gets activated, undergoing a conformational change, and binds to the damaged region, leading to the recruitment of the repair machinery [20]. During apoptosis, activated caspase-3 cleaves PARP, preventing DNA repair. Cleaved fragments of PARP easily be detected by western blotting and used as an indicator of apoptosis [21]. Litke JL et al examined the cytotoxicity of circular Broccoli expression through the cleavage of PARP in Western blots [22].

Anoikis is a form of apoptosis that results from a cell being detached from the extracellular matrix. Assays to measure this are particularly useful in cancer studies since cancer cells acquire the ability for anchorage-independent growth (leading to metastasis in in vivo systems) and become resistant to anoikis. There are, therefore, a number of assay systems available to assess anoikis. In these systems, cells are grown on plates coated with a matrix such as poly-HEME (poly(2-hydroxyethyl methacrylate)) that prevents cells from adhering. Cells are then co-stained using two fluorescent dyes. First, cell viability is determined with calcein-AM and monitored using fluorescent microscopy or a fluorescent plate reader with excitation at 485 nm and emission at 515 nm. Anoikis is concurrently assessed by the addition of a fluorescent DNA-binding dye such as ethidium homodimer (with excitation at 525 nm and emission 590 nm) that can only enter damaged cells. These dyes then fluoresce upon binding to DNA, allowing detection of anoikis quantitatively on a plate reader, or qualitatively using fluorescent microscopy. In addition to specialized kits, as long as cells are grown in suspension and prevented from attaching to a matrix, many authors use simple apoptosis assays such as DNA fragmentation or cleavage ELISAs to assess apoptosis [23]. Y-27632 at 10 µM can be used to inhibit anoikis [24].

Pyroptosis is a cell death pathway caused by bacterial infections that are mediated exclusively by caspase-1, which also leads to the activation of pro-inflammatory cytokines, including IL-1 and IL-18, in inflammasomes, likely at centrosomes in the case of NLRP3 and pyrin inflammasomes [25]. Although like necrosis, pyroptosis concurrently leads to membrane rupture and the release of inflammatory markers, this pathway can be distinguished by caspase-1 activation [26]. Since caspase-1 is not involved in other forms of cell death such as apoptosis [27], immunoblotting or ELISA for cleaved caspase-1 can assess pyroptosis simply and rapidly in isolated cell lysates. Gasdermin D (GSDMD), a pore-forming protein, plays a vital role in both NETosis and pyroptosis [28]. Samir P et al, for example, quantified pyroptosis based on SYTOX Green staining [29].

Parthanatos, also known as PARP-1 dependent cell death, is caused by the accumulation of PAR and the nuclear translocation of apoptosis-inducing factor (AIF) from mitochondria. Parkinson's disease involves parthanatos [30, 31].

Ferroptosis involves the accumulation of a lethal level of lipid hydroperoxides [32, 33]. GPX4, ACSL4, LPCAT3, (15-LOX and TFRC are known regulators of ferroptosis [34-36]. Chemicals, such as erastin, RSL3, and g GPX4 inhibitor ML162 [37] induce ferroptosis; ferrostatin-1 [37], liproxstatin-1, deferoxamine and zileuton inhibit ferroptosis [34-36]. Interestingly, selenoproteins, rare proteins present in all kingdoms of species with the 21st amino acid, selenocysteine, may be involved in preventing ferroptosis, ascribing the first specific role for selenium in vivo [38].

Necrosis is a form of cell death associated with acute cellular stress and can be caused by toxins, infection, or injury. Unlike apoptosis, this process is irreversible and is always detrimental to the cell and organism. During necrosis, the activation of cell surface receptors leads to a loss of plasma membrane integrity and an uncontrolled release of apoptotic proteins into the cell. This leads to widespread activation of the inflammatory response, preventing phagosomes from removing dead cells, and leading to a build-up of dead tissue. However, the distinction between the causes and effects of apoptosis and necrosis is complicated by reports describing pathogens that cause cell death with features of apoptosis (termed necroptosis [39, 40] ). Necrostatin-1 can be used to inhibit necroptosis [37].

As discussed above, apoptosis and necrosis play very different roles in cellular homeostasis and death. The easiest distinction to exploit experimentally is the induction of the inflammatory response, although notice must be taken that these are complex processes that may not always conform to simple traditional paradigms. Although assays such as trypan blue exclusion can be used to demonstrate membrane rupture, these assays provide no information regarding the pathway that led to the death of the cell. The best way to assess necrosis is, therefore, using the morphological changes that occur after a cell has died by light or electron microscopy (Figure 8). Specifically, necrotic cells exhibit nuclear swelling, chromatin flocculation, loss of nuclear basophilia, the breakdown of cytoplasmic structure and organelle function, and cytolysis by swelling, which leads to membrane rupture. However, in truth, apoptotic cells can also have plasma membrane rupture consistent with necrosis via a process called apoptotic necrosis. This occurs when apoptotic bodies have been formed in the absence of phagocytosis, which can lead to rupture of the bodies. It is therefore difficult to determine how a necrotic cell actually died.

This section is provided by Labome to help guide researchers to identify most suited cell-based assay kits. Labome surveys formal publications. Table 3 lists the major suppliers for reagents/kits used in detecting apoptosis in the Labome survey. For example, Nam S et al measured the apoptotic cells in 3D hydrogels with a TUNEL kit from Thermo Fisher Scientific [41]. Maya-Ramos L and Mikawa T detected apoptotic cells during chick embryo gastrulation with the In Situ Cell Death Detection Kit from Roche (11 684 817 910) [42].

This article is derived from an earlier version of an article authored by Dr. Laura Cobb "Cell-Based Assays: the Cell Cycle, Cell Proliferation and Cell Death", written in February 2013.