The fact that flow cytometry enables simultaneous measurement of multiple cellular parameters is one of the most powerful aspects of this technology. The technique has a wide range of applications including the identification of antigens within or on the surface of a cell, DNA and RNA analysis and a number of other potential uses. Some examples of the parameters that can be assessed using flow cytometry are described below.
Flow cytometry can be used to evaluate changes that occur in cells over a short period of time. Biological activities such as variation in calcium content in response to drugs; the generation of reactive oxygen species or mitochondrial membrane changes during apoptosis and phagocytosis rates using labelled bacteria, can all be assessed using flow cytometry.
A DNA binding dye can be added to a cell population to assess cell viability after the introduction of a pathogenic organism. When the integrity of the plasma membrane is compromised, a cell becomes necrotic and allows viability dyes to enter it. Dead cells can now be identified at the early or late stages of necrosis using a range of viability dyes, DNA quantification and through measuring the side scatter of the membrane probe Bis-oxonal.
Self destruction of a cell as instructed by genes is referred to as apoptosis. Flow cytometry can be used to detect the characteristic morphological and biochemical changes that occur during apoptosis. Examples of morphological changes include changes in cell shape, loss of structures on the cell surface, cell detachment, condensation of the cytoplasm, cell shrinkage, phagocytosis of cellular residues and changes in the nuclear envelope. Some examples of biochemical changes include proteolysis, DNA denaturation, cell dehydration, protein cross-linking, and a rise in the free calcium ions.
Necrosis measurements can be derived from oncosis, apoptosis and self phagocytosis processes. Oncosis refers to necrosis as the result of an event that causes the cell to swell rather than shrink (as occurs in apoptosis). During oncosis, the cell and the organelles inside it swell. The plasma membrane eventually ruptures and is followed by the necrotic stage where the cell releases proteolytic enzymes that damage surrounding tissue. The cell shrinks (apoptosis) and changes occur in the mitochondrial structure and transmembrane potential. The chromatin also condenses.
Cell cycle analysis
Cellular proliferation can be assessed by measuring the different stages of the cell cycle in a population of cells. The DNA content varies with each phase of the cell cycle and this can be assessed using fluorescent DNA binding dyes and monoclonal antibodies to detect the expression of antigens. As histone H3 undergoes phosphorylation between prophase and anaphase, this protein can be used to distinguish between G2 and m phases when assessing the cell cycle.
Some other examples of the parameters that can be assessed using flow cytometry are listed below:
- Measurement of cell pigments such as chlorophyll or phycoerythrin
- Measurement of the DNA copy number variation using Flow-FISH (Fluorescent in-situ hybridization) or BACs-on-Beads technology
- Measurement of intracellular antigens such as cytokines or secondary mediators
- Measurement of enzymatic activity
- Measurement of oxidative burst
- Detection of glutathione
- Measurement of cell adherence