Apoptosis

Apoptosis is a form of cell death characterized by several features including cell shrinkage, membrane blebbing, chromosome condensation, nuclear fragmentation, DNA laddering, and the eventual engulfment of the cell by phagosomes. It maintains a balance between cell death and proliferation in a cell population, regulating the total number of cells²². Apoptosis also plays vital roles in the immune system²³ and some developmental processes.

Dysfunctions in apoptosis can lead to disease by causing excessive cell death, such as in abnormal development and degenerative diseases, or by allowing too many cells to survive, an imbalance in the opposite direction known to cause cancer and allow viral infections to persist. To further understand these disease areas and work towards effective therapies in these fields, identifying and exploring the role of apoptosis is a fundamental first step²⁴.

Using apoptosis assays as part of your analysis can help to identify early indicators of cell health changes. You can assay apoptosis using a number of different approaches. As with cell viability assays, the most appropriate apoptosis assay will depend on factors such as your sample type and number of cells. It is often best to combine more than one assay.

Apoptosis occurs via a complex signaling cascade. Figure 17 shows the main stages of apoptosis and the approximate relative time when markers for those events are likely to be detected.

These stages do not happen in a sequential order, and many of them will overlap and occur at the same time.

As cell death can occur by several different pathways, including apoptosis, necrosis, autophagy, and necroptosis, some of which share characteristics, you may need to examine multiple apoptosis markers to confirm that this is the mechanism of cell death in your experimental system.

The table below shows the main apoptosis markers and the most common methods used to study them.

You can determine how cells are dying by measuring markers that are activated in different types, and at different stages, of cell death.

There are a number of methods for running an apoptosis assay to measure these markers of apoptosis.

Loss of membrane asymmetry in apoptosis can be detected using Annexin V. Annexin V binds to phosphatidylserine, which migrates to the outer plasma membrane in apoptosis. Analysis is typically by flow cytometry. Pair Annexin V with a membrane-impermeable dye like 7-AAD to distinguish between intact, apoptotic, and necrotic cells.

Advantages:

• Suitable for in-vivo, tissue and cell culture studies
• Allows for earlier detection of apoptosis than TUNEL²⁵

Disadvantages:

• Not suitable for use in fixed cells

Chromatin condensation and genomic DNA fragmentation, together with cell membrane blebbing, are considered morphological hallmarks of the terminal stages of apoptosis.

Chromatin condensation:

During apoptosis, chromatin undergoes a phase change from a heterogeneous, genetically active network to an inert, highly condensed form. When stained with DNA-binding nuclear dyes, the compacted chromatin will be brighter than the chromatin from non-apoptotic cells, and the condensed nuclei can be easily identified by fluorescence microscopy (qualitative detection) and/or flow cytometry (quantitative detection).

Genomic DNA fragmentation:

Condensed chromatin can be fragmented by a specific nuclease called Caspase-Activated DNase (CAD). Activation of CAD by the caspase cascade leads to specific cleavage of the DNA at the internucleosomal linker sites between the nucleosomes, generating fragments of ~ 200 base pairs known as DNA ladders.

A classical method to detect DNA ladders is to examine fragmented genomic DNA on an agarose gel. This is a simple, semi-quantitative method that provides a robust answer. Review the protocol  to learn more.

An alternative method for detecting DNA fragmentation involves the identification of nicks (or strand breaks) using a TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay.

The TUNEL staining / TUNEL assay method relies on the enzyme terminal deoxynucleotide transferase (TdT), which attaches deoxynucleotides to the 3’-hydroxyl terminus of DNA breaks. TdT is expressed in certain immune cells and acts during V(D)J recombination – the process that generates antibody diversity.

In TUNEL staining, the nucleotides attached by TdT are tagged either directly with a fluorescent label or with a chemical label that can be indirectly linked to either a fluorescent label or an enzyme.TUNEL staining is a modern alternative to analyzing the formation of DNA fragments during apoptosis using agarose gel electrophoresis.

TUNEL staining / the TUNEL assay is most commonly analyzed by light microscopy. Fluorescent TUNEL staining / TUNEL assay methods are also suitable for analysis by flow cytometry.

See our TUNEL staining / TUNEL assay guide for more details.

Advantages:

• TUNEL assay has high sensitivity and widely used
• Detection of DNA ladders by gel electrophoresis is simple and robust

Disadvantages:

• Risk of false positives with TUNEL assay

An increase in the percentage of cells stalled at G1 is another consequence of DNA fragmentation and can be readily detected with a flow cytometer. Once cells are permeabilized with, for example, a 70% ethanol solution, the DNA fragments will leak out and create a population of cells with reduced DNA content. When cells are stained with a DNA staining dye such as propidium iodide, a DNA profile representing cells in the different stages of the cell cycle (G1, S-phase and G2M) can be observed by flow cytometry. The apoptotic cells are easily identified as the subG1 population, seen to the left of the G1 peak. The subG1 fraction will include all the dead cells in the population regardless of the type of cell and therefore this parameter on its own is not a good indicator of apoptosis.

Advantages:

• Compatible with cells of any species that can be prepared as a single-cell suspension

Disadvantages:

• Not a good indicator of apoptosis on its own
• Requires fixation

Together with DNA fragmentation, the final execution phase of apoptosis is characterized by dynamic membrane blebbing and cell contraction. During apoptosis, the cell cytoskeleton breaks up, causing some parts of the cell membrane to bulge outwards. The bulges eventually separate from the cell taking a portion of the cytoplasm with them and forming what are known as apoptotic bodies.

Membrane blebbing can be observed in live cells using phase-contrast microscopy. If you are not able to use live cells or would like to use cells that you have prepared for studying other parameters (for example, cells harvested for DNA fragmentation quantification or chromatin condensation), you can detect caspase substrates associated with apoptotic membrane blebbing. Be aware, however, that this is an indirect method and it may give you false positive/negative results.

Caspase substrates associated with apoptotic membrane blebbing include:

• Gelsolin
• ROCK-1 kinase
• P21-activated kinase (PAK)

Advantages:

• Enables real-time analysis of apoptosis within a population²⁷

Disadvantages:

• Risk of false positive/negative results with caspase substrates detection method

Caspases are a family of conserved cysteine proteases that play an essential role in apoptosis.

Mammalian caspases can be subdivided into three functional groups: initiator caspases (caspase 2, 8, 9 and 10), executioner caspases (caspase 3, 6 and 7), and inflammatory caspases (caspase 1, 4, 5, 11 and 12). Initiator caspases initiate the apoptosis signal while the executioner caspases carry out the mass proteolysis that leads to apoptosis. Inflammatory caspases do not function in apoptosis but are rather involved in inflammatory cytokine signaling and other types of cell death such as pyroptosis.

Initially synthesized as inactive pro-caspases, caspases become rapidly cleaved and activated in response to granzyme B, death receptors and apoptosome stimuli. Caspases will then cleave a range of substrates, including downstream caspases, nuclear proteins, plasma membrane proteins and mitochondrial proteins, ultimately leading to cell death.

Activated caspases can be detected using antibodies with IHC, western blotting, or flow cytometry. Caspase activity assays either use peptide substrates, which are cleaved by caspases in cell extracts, or similar substrates that bind to activated caspases in live cells. Caspase specificity varies by substrate.

The answer provided by different experimental methods will confirm whether one or more specific caspases are active or inactive. It is always best practice to use more than one method to confirm specific caspase activation.

Advantages:

• Quick and convenient for determining caspase activation

Disadvantages:

• Caspase activation can occur in non-apoptotic events²⁸
• Cleavage specificities of caspases overlap, therefore reliance on a single substrate/assay is not recommended for identification of a specific caspase

Usually located in the space between the inner and outer mitochondrial membranes, cytochrome C is released into the cytoplasm following total loss of mitochondrial membrane potential (ΔΨm).

The collapse of the ΔΨm is a catastrophic event. It leads to the opening of the mitochondrial permeability transition pores in the mitochondrial membrane, and the subsequent release of cytochrome C in the cytosol, which in turn triggers other downstream events in the apoptotic cascade.

Once the mitochondrial pores are opened and cytochrome C is released, the apoptotic cascade reaches a “point of no return” from which it is very unlikely that the cell can recover, and death is the most likely outcome.

The most common technique to detect cytochrome C release is through western blot on protein extracted from different subcellular compartments. It is very important in this case to ensure that the different subcellular fractions are not contaminated with other fractions. This can be easily checked with specific and reliable subcellular markers:

• Cytoplasmic markers: GAPDH, actin
• Mitochondrial markers: VDAC1, PDH-E1

On the other hand, immunofluorescence staining of fixed cells with a cytochrome C antibody at selected time points can be used to visualize cytochrome C release from the mitochondria into the cytoplasm.

Advantages:

• Enables analysis in living or fixed cells

Disadvantages:

• Accurate quantitative analysis is difficult with western blot²⁹

Dyes that accumulate in mitochondria due to the mitochondrial membrane potential are also used in the analysis of apoptosis. During apoptosis, several changes happen in the mitochondria, most notably the loss of mitochondrial membrane potential.³⁰

For more information, see the earlier section on metabolism-based assays. Apoptotic cells stain more weakly with these dyes due to the loss of membrane potential.

Glutathione (GSH) is a tripeptide which prevents cell damage caused by reactive oxygen species such as free radicals and peroxides. The monitoring of reduced and oxidized GSH in samples is essential for evaluating the redox and detoxification status of the cells and tissues against oxidative and free radicals mediated cell injury. The ratio of oxidised glutathione (GSSG) to reduced glutathione (GSH) is associated with regulation of apoptosis, and as a result, an imbalance towards increased GSSG can lead to cell death.³¹

Glutathione assays allow detection of total glutathione changes during cellular response to toxicity, apoptosis and other conditions.

Advantages:

• Simple
• Can be used together with other reagents, such as 7-AAD for multi-parametric study of cell viability and apoptosis

Disadvantages:

• Assay components have short stability at room temperature, so assay must be performed quickly

Other assays are used to assess necrosis, anoikis and autophagy. Learn more about the mechanisms of apoptosis and other forms of cell death in our three comprehensive guides to apoptosis , necroptosis , and autophagy.