Dysregulated apoptosis is implicated in conditions that include cancer, neurodegenerative disease, and a broad range of autoimmune disorders. Understanding the mechanisms of apoptosis therefore promises to guide the development of effective treatments. This article provides a brief overview of apoptosis before describing some common apoptosis detection assays. It also highlights the advantages of using calcium-independent apoptosis probes in place of Annexin V, which has historically been considered a gold standard for apoptosis detection.
Apoptosis, also known as programmed cell death, is critical to normal development. For example, in the developing nervous system, apoptosis helps delineate cell populations in the brain and spinal cord by controlling cell numbers. Apoptosis also safeguards physiological functions of adult tissues and provides an essential mechanism of defense through homeostatic regulation of cell populations and elimination of cells that have been damaged by infection or toxins, respectively.
Early-stage, mid-stage, late-stage apoptosis
The main difference between apoptosis and necrosis (a form of cell death where cells typically swell and burst due to injury) is that apoptosis is a tightly controlled process that culminates in cell engulfment via phagocytosis. As well as preventing the release of cellular contents into the external milieu to avoid triggering a potentially harmful inflammatory response, apoptosis provides opportunities for many cellular components to be recycled.
During apoptosis, cells display several key morphological characteristics that can be exploited for apoptosis detection. These include cell shrinkage, cytoskeleton collapse, destruction of the nucleus and its contents (karyorrhexis), plasma membrane blebbing, and budding of the cell to form small apoptotic bodies. The expression and activity of various proteins, including many enzymes, is also altered throughout apoptosis; monitoring these changes alongside assessing cellular morphology helps track apoptotic onset and progression.
Included among the many different assays available for apoptosis detection, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining is an established method that involves attaching fluorophore- or enzyme-labeled deoxynucleotides to the 3’ ends of DNA fragments produced as a result of endonuclease action. This enables visualization of DNA fragments by microscopy or can be adapted to support flow cytometry-based analysis of late-stage apoptosis.
An alternative and equally popular approach is to detect cleaved caspase-3 in apoptotic cells by techniques such as microscopy, flow cytometry, or ELISA. In healthy cells, caspase-3 exists as an inactive pro-enzyme. However, following its activation through cleavage by activated initiator caspases, caspase-3 (an effector caspase) cleaves multiple structural and regulatory proteins that are crucial for cell survival and maintenance.
Another widely used method for apoptosis detection relies on labeled Annexin V for staining phosphatidylserine (PS), a protein normally found at the inner surface of the plasma membrane. During apoptosis, disruption of plasma membrane asymmetry leads to translocation of PS and its exposure at the cell surface, where it functions to promote cell engulfment by phagocytes. Exposed PS can be detected by flow cytometry, microscopy, or ELISA as an early marker of apoptosis.
An important advantage of Annexin V staining is that it does not require cells to be fixed and permeabilized prior to staining. Not only does elimination of these protocol steps shorten experimental workflows, but it also helps prevent other cell surface markers (e.g., those used for cellular identification) from being damaged by harsh fixation and permeabilization conditions.
Counterbalancing these benefits, a main drawback of using Annexin V for apoptosis detection is the need to include a high calcium concentration (<2mM) in the staining buffer. This can lead to artefactual cell death and activation, meaning that probes capable of binding apoptotic cells independently of Ca2+ ion presence are often a preferred choice.
Probes able to detect apoptosis without causing unwanted cytotoxic effects promise improved data quality, not least by reducing background staining that can mask detection of the target of interest. Specifically, probes designed for apoptotic cell detection in standard (calcium-free) flow cytometry staining buffer are seeing increased use across a diverse array of research areas.
Apotracker™ is a family of fluorogenic probes that binds apoptotic cells independently of Ca2+. Because it exhibits a linear relationship with Annexin V staining, it has been suggested to detect externalized PS residues. While both Annexin V and Apotracker™ are readily compatible with flow cytometry-based studies, Apotracker™ can also be incorporated into microscopy applications using live cells to increase confidence in results.
Five-day old HeLa cells were stained with Calcein Red-AM (an indicator of live, healthy cells), Helix NP™ Blue, and Apotracker™ Green (Apo-15).
BioLegend offers a broad selection of reagents for apoptosis detection including Apotracker™ probes that can be used for both flow cytometry and microscopy-based applications. To learn more, visit biolegend.com