Apoptosis is a carefully regulated process that can be influenced by many factors, and thus tracking and monitoring these processes is a vital and regular part of biological research. Flow cytometry is a high-throughput method of cell counting and sorting, and is used to quickly determine the rate of apoptosed cells in a sample following, for example, exposure to a drug lead under investigation.
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As a spectroscopic technique, flow cytometry is capable of inferring the reason for apoptosis by detecting caspase activity, DNA cleavage, membrane dynamics, and other parameters by cell pre-staining with dyes or fluorophores.
As a high-throughput method flow cytometry can analyze 10,000 cells in minutes or seconds, allowing numerous parameters to be assessed across a range of cell lines in an optimized and cost-effective manner.
Apoptosis reporting
There are two main routes of apoptosis: the extrinsic pathway via caspase 8, and the intrinsic pathway by caspase 9. Once activated these caspases initiate a cascade that results in the activation of additional caspases that cause DNA fragmentation, protein degradation and cross-linking and expression of receptors that signal for phagocytosis.
Numerous apoptosis reporter tags have been developed that can bind with the caspases, or any of the many other elements expressed during apoptosis, and thereby indicate the state and stage of apoptosis in a cell. The initiator caspases 8 and 9 are particularly useful to indicate the cause of apoptosis, and other indicators such as membrane phospholipid asymmetry and DNA fragmentation can also indicate the route and progress of apoptosis.
Modern proliferation kits provide fluorescent dyes that stain the mitochondria, meaning that daughter cells conserve around half of the signal of the parent, allowing the generation to be tracked.
Combining multiple fluorescent tags of distinct wavelengths in this way allows a significant quantity of information to be derived from a single sample using high throughput flow cytometry.
Following the application of a toxin, drug, or therapy under investigation, the stage and method of selectively induced apoptosis in a mixed cell population can be determined, with further information regarding how exposure may have affected cell dynamics and division.
Impedance flow cytometry
Besides the detection of dyes by spectroscopic methods flow cytometry can also gain information on the cells under investigation by impedance measurements, requiring no preparatory staining and avoiding perturbation of the biological system.
At low frequencies (105 Hz) the cell membrane is polarized and thus acts as an insulator, allowing researchers to infer the volume of the cell. The cell membrane is less polarized to medium frequencies(106 Hz), which allows cell membrane integrity to be assessed, while high frequencies (107 Hz) pass through the membrane allowing the interior of the cell to be examined.
Cells undergoing apoptosis or necrosis have been identified by impedance measurements, inferred by comparative cell size and cell membrane characteristics.
In the future, higher frequency impedance lab-on-a-chip devices will be available that are able to characterize internal organelle state and position, allowing more details regarding the route of apoptosis to be inferred. Combining impedance measurements with tagged specimens will allow a greater number of parameters to be assessed simultaneously.
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