Apoptosis is a dynamic, multi-step, programmed cell death process. Improved understanding offers the promise of preventing cell death when inappropriate (such as in neurodegenerative disease), and triggering it when the body is invaded by rogue cancer cells.
By introducing flow cytometry into the mix, scientists can follow apoptotic progression from induction through surface receptors all the way to the very end stages of DNA fragmentation, oftentimes simultaneously (important, since apoptosis is not defined by a single parameter). And this can be done fairly rapidly. Sorting yields a clean population of cells, as opposed to measuring across a heterogeneous population in different phases (as is the case with assays like western blot) leading to more accurate and reproducible experimental results.
This approach also offers flexibility, and quantitative, cell type specific, and single cell data, says Lori Roberts, director of bioscience at Biotium. Further analysis can be performed downstream on sorted cell populations. “Flow cytometry allows scientists to correlate an individual cell’s apoptosis status with other features, like surface or intracellular markers,” adds Ferogh Ahmadi, senior product manager of research reagents for BD Biosciences. This is especially useful when examining mixed origin cell samples, such as is found in tumors or blood.
Below is a description of the more common assays that can be used with flow cytometry.
Annexin V Assay: The flipping of phosphatidylserine (PS), from the inner to the outer leaflet of the cell membrane is an early to mid phase signal of apoptosis. This assay relies on Annexin V binding to exposed PS at the cell surface.
Performed on living cells, this assay quickly evaluates apoptotic status. It is often paired with a membrane impermeable dye. “Typically cells are double-labeled with Annexin V and an impermeable DNA dye that only labels dead cells with a compromised membrane. Early apoptosis is identified by the presence of the Annexin V label and absence of dead cell marker. Late apoptosis or necrosis will have both labels,” says Martha O’Brien, senior research scientist at Promega.
Image: Annexin V and Propidium Iodide Staining in CD95-Treated Mouse Thymocytes. Balb/c thymocytes were cultures with (right plot) or without (left plot) 10 μg/mL Purified NA/LE Hamster Anti-House CD95 antibody (clone Jo2) for 4 hours. Cells were harvested, stained with BD Horizon™ BV421 Annexin V and BD Pharmingen™ Propidium Iodide Staining Solution (PI), and analyzed by flow cytometry using a BD LSRFortessa™ X-20 Cell Analyzer System. CD95-treated cells show an increase in apoptotic (Annexin V+, PI-), late apoptotic/early necrotic (Annexin V+, PI+), and late necrotic (Annexin V-, PI+) cells. Image courtesy of BD.
Both BD and Biotium offer a wide range of fluorochrome choices for Annexin V conjugates, which allows for maximum flexibility when incorporating this assay into multicolor panels.
Do note that although normally considered an indicator of apoptosis, flipping of PS is reversible until the depolarization of the mitochondrial membrane, highlighting the importance of multiple assays to confirm cell death.
Mitochondrial Membrane Potential: Loss of mitochondrial membrane potential is another easily assayed feature. This occurs via the intrinsic pathway when Bcl-2 family member proteins insert into the cell membrane and create pores causing the collapse of the electrochemical gradient across the mitochondrial membrane. Once this happens, cells are committed to apoptosis.
Potentiometric dyes detect reduced mitochondrial potential. This reads as reduced fluorescence as the dye, which is normally attracted to the negative charge of the mitochondria, disperses once the membrane potential collapses.
To monitor mitochondrial changes, Biotium offers MitoView™ 633 mitochondrial membrane potential dye. BD’s MitoScreen kits and Mitostatus dyes are also available.
Caspases: Specific or pan-caspase activity can be detected by directly inhibiting caspase activity or via antibody detection. Analysis can be as simple as whether or not caspases are present to the more complex deciphering of pathways and downstream targets.
For example, Ahmadi explains, “Caspase-3 is an executioner protein activated in both the intrinsic and extrinsic apoptotic pathways. It can be detected both by antibodies specific for the active form of caspase-3 or fluorescent caspase inhibitors that bind to caspases in their active form only.”
Fluorochrome inhibitor of caspases (FLICA) reagents pass through the plasma membrane and bind covalently to active caspases. The fluorescent signal is a measure of the amount of caspase activity in the living cell. Viability dyes should be used in conjunction to distinguish between apoptotic and dead cells.
Caspase activity can be monitored with BD’s Live Cell Caspase probes or caspase antibodies.
Biotium’s NucView® Caspase-3 Substrates work similarly to FLICA assays, but do not prevent caspase activity, thus allowing for the monitoring of caspase activity in real time. These kits are also available with Annexin V or late apoptosis and necrotic markers.
Image: Apoptotic HeLa cells stained with NucView® 488 Caspase-3 Substrate and CF®594 Annexin V. NucView 488 stains DNA in apoptotic cells with green fluorescence after cleavage by caspases in apoptotic cells, while CF594 Annexin V stains phosphatidylserine exposed on the surface of apoptotic cells wth red fluorescence. Image courtesy of Biotium.
Late Apoptosis Assays: Both light scatter properties and DNA fragmentation can be observed with flow cytometry. “Apoptotic cells tend to shrink and eventually bleb, producing small, membrane-bound particles containing a portion of the cytoplasm and cellular components that are eventually consumed by phagocytes,” says Ahmadi. Both of these features can be detected by scatter. Cell shrinkage leads to forward scatter decrease. Blebs, which are very small, have both low forward and side scatter.
A couple of caveats—light scatter analysis does not give any information about the underlying apoptotic mechanism. Also, if cells have not lost enough DNA, apoptotic cells with be indistinguishable from living G1/G0 cells.
“Toward the end of apoptosis, nuclear DNA is condensed and then degraded. This results in double-stranded DNA breaks, which can be detected using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays. These assays incorporate nucleoside analogs into double-strand breaks, allowing their detection by flow cytometry or other fluorescence assays,” explains Ahmadi.
Tips, tricks, and the fine print
These assays are relatively simple and “stackable.” But apoptosis is dynamic and sensitive to techniques and external factors. Ahmadi suggests using multiple time points in case certain features are not detected within a specific timeframe.
“It is very important to note that depending on the mechanism or stage, apoptotic cells can display certain features in common with necrotic cells. Therefore, determining which pathway cells are following may require assaying for multiple features of each pathway,” says Ahmadi. To distinguish between late-phase apoptosis and necrosis, he suggests assaying differences between the two processes, such as the presence of caspase activity, or utilize inhibitors specific to each pathway. Pan-caspase inhibitors can be used to inhibit caspase activity in apoptosing cells.
The type of cells being used may also influence your decision about how to best assess apoptosis. Not surprisingly, Lissette Wilensky, senior scientist at BD, says that suspension cells are the easiest to adapt to flow cytometry. Adherent cells can also be used, but trade the trypsin for non-enzymatic dissociation buffers. Although trypsin works quickly, it “can lead to artifacts from cleaved cell surface proteins,” says Wilensky. Roberts adds that trypsin can cleave off cell surface markers, too. Depending on the target, scientists may want to stain cells before detaching them (detached cells may internalize surface proteins).
When using adherent lines, Roberts says to take care to collect any floating cells for viability analysis since dead cells often detach from the substrate. And pay attention to your technique. “Many adherent cells also require attachment for survival, so they cannot be kept in suspension for very long before analysis, or viability could be affected.”
As with all experiments, proper controls are a must, as is a thorough understanding of the filters, lasers, and capabilities of the cytometer. Cell death often causes increased autofluorescence, and dead cells can bind antibodies indiscriminately. According to Wilensky, “To account for differences in healthy and unhealthy cells, it is often helpful to create a positive control by treatment with an apoptosis inducer to allow the investigator to establish expected changes in autofluorescence and other apoptosis-associated properties for their cell type of interest.”
Finally, know when another protocol is more appropriate. The target will inform the approach. “Western blotting is often used to detect cleavage of caspases or apoptosis target proteins like Bcl-2 family members, where the antibody can't distinguish cleaved or uncleaved protein unless the proteins are first separated on a gel,” says Roberts. Western blotting can also measure protein interactions.
When it comes to utilizing flow cytometry for apoptosis, Wilensky says that although the tools have remained largely the same, the ability to multiplex probes has become easier. “Instrumentation with more lasers and more fluorescent channels has made it easier to spread out fluorochromes, thus facilitating straightforward panel design with easier compensation.”