Monitoring Apoptosis by Flow Cytometry

Apoptosis, also known as programmed cell death, is crucial for maintaining healthy cell population and serves as a natural “cleansing” process. Dysfunction in apoptosis is linked to the uncontrolled growth of cancer cells, for example. Discriminating apoptotic from normal cells is an important task for studying apoptosis, but there isn’t just one test. Apoptosis is a complex, multistep process that should be assayed and monitored at different stages to gain a better understanding of how it works. This is where methodologies like flow cytometry have enabled researchers to better dissect and monitor the progression of apoptosis in cells. A key feature of flow cytometry assays is the ability to measure multiple parameters simultaneously. Here we discuss a series of assays commonly used for monitoring apoptotic cells by flow cytometry.

Monitoring early-stage apoptosis

Early detection of the onset of apoptosis provides researchers with insight into how to regulate or possibly control the process. Tool providers have created a variety of assays that scientists can use to monitor early-stage apoptosis events. A change in mitochrondrial-membrane potential is one of the earliest indicators of apoptosis. Active mitochondria have negatively charged interiors, so positively charged dyes accumulate within them. Thermo Fisher Scientific’s MitoProbe™ JC-1 accumulates in mitochondria, and its emission wavelength changes with mitochondrial-membrane potential. This results in a fluorescence shift from red to green, as the mitochondrial-membrane potential declines with the onset of apoptosis. BioVision’s MitoCapture™ Apoptosis Detection Kit also uses a cationic dye that fluoresces red in mitochondria in healthy cells—but remains in the cytoplasm and fluoresces green in apoptotic cells because of the disruption of the mitochondrial-membrane potential. Other assays include Abcam’s TMRE-Mitochondrial Membrane Potential Assay Kit, Bio-Rad Laboratories’ Mitochondrial Membrane Potential and Mitochondrial Permeability Transition Kits and R&D Systems’ DePsipher™ Kit.

Assays for intermediate-stage apoptosis

Assays for membrane asymmetry

During the early to intermediate stage of apoptosis, phosphatidylserine molecules translocate from the inner to the outer layers of the cell membrane. Because of this event, Annexin V, which binds to phosphatidylserine, has become a key marker for apoptosis. In the outer layer of the cell membrane, phosphatidylserine is accessible to the outside world and hence binding by fluorescently labeled Annexin V. “By combining [Annexin V] kits with any viability dye, such as propidium iodide, apoptotic cells can be distinguished from necrotic cells,” says Mike Blundell, product manager in the Life Science Group at Bio-Rad Laboratories. Kits for this assay are also available from Abcam, BD Biosciences, BioLegend, Biovision, Cell Signaling Technology, eBioscience, MBL International, MilliporeSigma, R&D Systems, Santa Cruz Biotechnology and Thermo Fisher Scientific.

Bio-Rad’s pSIVA™ Flow Cytometry Kit can additionally assess early membrane asymmetry. The pSIVA probe fluoresces green when it binds phosphatidylserine in the presence of Ca²⁺. “At this point, apoptosis can be reversible and is before the mitochondrial outer membrane permeabilization, which is considered to be the point of no return,” says Blundell. An alternative tool is a dye that fluoresces when phosphatidylserine translocates, such as Thermo Fisher’s Violet Ratiometric Membrane Asymmetry Probe/Dead Cell Apoptosis Kit.

Caspase-based assays

In healthy cells, caspases exist in the inactive state designated as pro-caspases, which are converted to active caspases by proteolysis. This proteolytic event is an important milestone in apoptotic signaling cascades. Cleavage-specific antibodies are offered by Cell Signaling Technology for the activation of several types of caspase proteins, as well as PARP (poly ADP-ribose polymerase), another enzyme involved in apoptosis. “Antibodies enable quantification of the amount of protein and/or modified protein per cell in addition to the quantification of the number of responding cells in a population,” says Christopher Manning, group leader in flow cytometry at Cell Signaling Technology.

A common caspase-based assay uses the cell-permeant reagent FLICA (fluorochrome inhibitor of caspases), which consists of a caspase inhibitor sequence linked to a fluorescent tag. FLICA molecules bind to active caspases, and the fluorescent signal is proportional to the level of caspase activity. Bio-Rad offers FLICA Caspase Kits for caspases 1, 2, 3/7, 8, 9, 10 and a Poly Caspase Kit for all types. Promega’s CaspACE^TM FITC-VAD-FMK In Situ Marker, which binds to any activated caspase, often is used as a marker for apoptotic cells in flow cytometry, says Martha O’Brien, senior research scientist at Promega.

Vergent Bioscience’s CAS-MAP^TM Caspase Apoptosis Assay Probes are available in pan-caspase form or selective for caspase 3/7 or caspase 8. The probes can be used to label cells either in vitro or in vivo for subsequent flow cytometry. “Apoptotic cells can be labeled in vivo by injection of our CAS-MAP^TM Caspase Probes,” says Eric Bensen, director of research and development at Vergent Bioscience. “Tissues can be removed, digested to single cells and analyzed by flow cytometry.” FLICA assays for active caspases are also available from Aviva Systems Biology, Gentaur, Novus Biologicals and Thermo Fisher Scientific, among others.

Assays for late-stage apoptosis

The end of apoptosis is characterized by changes in the cell’s nucleus, such as the condensation of chromatin and fragmentation of DNA. Apoptotic cells have smaller nuclei when visualized with UV-sensitive stains. Abcam, Enzo Life Sciences, Thermo Fisher Scientific and several other vendors offer chromatin condensation assays.

Assays for DNA fragmentation, known as TUNEL (terminal deoxynucleotidyl transferase-dUTP nick end labeling) assays, are offered by many vendors, including Bio-Rad Laboratories, Biovision, MilliporeSigma, Promega, R&D Systems and Thermo Fisher Scientific. O’Brien says Promega’s DeadEnd^TM Fluorometric TUNEL System frequently is used to monitor later apoptosis. In general, though, TUNEL assays should be used along with another assay to verify apoptosis. “Fragmented or degraded DNA can occur for reasons other than apoptosis, so any TUNEL assay has to be used with good controls to ensure that the fragmented DNA is due to apoptosis,” says O’Brien. “Combining a TUNEL assay with another marker of apoptosis is an excellent way to provide certainty of the biology.”

Fluorescently labeled antibodies for apoptotic markers are another helpful tool for monitoring apoptotic cells. “Antibodies enable researchers to identify the key regulators of apoptosis in various cells or tissues and to understand where in the cascade a change is being affected by a treatment,” says Manning. Bio-Rad, Cell Signaling Technology, Promega and others offer a range of antibodies for detection of apoptotic cells by flow cytometry. With such a broad toolkit—one covering many stages of apoptosis—researchers have many options for uncovering the secrets of what happens to cells when it’s their time to go.