Flow cytometry is used to determine the physical and chemical properties of cells in a heterogeneous population. Using this method, multiple parameters of single cells can be analyzed simultaneously. This article describes the basic principles of flow cytometry, it's applications and how cytometric data can be analyzed.
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Basic principles of flow cytometry
Flow cytometry is used when there is a need to profile a large number of different cell types in a population. The cells are separated on the basis of differences in size and morphology. Additionally, fluorescently-tagged antibodies that target specific antigens on the cell surface can be used to identify and segregate various sub-populations.
The basic steps include passing the cells through a narrow channel, such that each cell is illuminated by a laser one at a time. A series of sensors then detect the refracted or emitted light, and this data is integrated and compiled to generate information about the sample.
What can flow cytometry be used to measure?
Flow cytometry helps to analyze several parameters of a cell simultaneously. Some of these parameters are described below:
Functional analysis
This method can determine biological activity inside cells, such as the generation of reactive oxygen species, mitochondrial membrane changes during apoptosis, phagocytosis rates in labelled bacteria, native calcium content, and changing metal content in response response to drugs, etc.
Determining cell viability
This method can also be used to assess cell viability after the addition of pathogenic organisms or drugs. Any breach in cell membrane integrity can be determined using dyes that can enter the punctured cell membrane. Fluorescent probes such as bis-oxonol can bind to proteins present on the cell membrane, allowing for the identification of various stages of necrosis.
Measuring apoptosis and necrosis
Apoptosis or programmed cell death is accompanied by characteristic changes in cell shape, loss of structures, cell detachment, condensation of the cytoplasm, cell shrinkage, phagocytosis of cellular residues and changes in the nuclear envelope.
Some of the biochemical changes include proteolysis, DNA denaturation, cell dehydration, protein cross-linking, and a rise in the free calcium ions. These physical and biochemical changes can be detected using flow cytometry.
Oncosis is a necrotic event where the cell starts to swell rather than shrink. This leads to rupture of the plasma membrane and release of proteolytic enzymes that can also damage the surrounding tissues. These changes in the plasma membrane and cell shape can be assessed using flow cytometry.
Cell cycle analysis
The amount of DNA present in the nucleus varies during each phase of the cell cycle. This variation in DNA content can be assessed using fluorescent dyes that bind to DNA or monoclonal antibodies, which can allow the detection of antigen expression.
Other factors including the content of cell pigments such as chlorophyll, DNA copy number variation, intracellular antigens, enzymatic activity, oxidative bursts, glutathione, and cell adherence can similarly be measured using this method.
How do flow cytometers work?
Fluidics
During flow cytometry, a sheath fluid hydrodynamically focusses the cell suspension through a small nozzle such that only one cell passes the laser light at a time. A detector is placed in front of the laser beam such that it can capture the forward scattered light from the cells, while several detectors are also placed to the sides to measure the amount and intensity of light scattered in each direction.
Forward scatter refers to the light refracted by a cell that is traveling in the same direction as it was traveling originally. The proportion of light that is forward scattered is correlated with the cell size, where larger-sized particles produce more forward scattered light.
Side scatter refers to the refracted light that is orthogonal to the direction of the light path. Side scattered light provides information about the granularity, where the highly granular cells produce more side-scattered light compared to cells with low granularity.
For example, while performing flow cytometry of blood samples, large and granular granulocytes show a high forward and side scatter; monocytes that are large but not granular show a high forward but lower side scatter. Thus, based on the proportion of light that is forward and side scattered, different types of populations can be separated.
Separation based on fluorescence emission
Apart from forward and side scatter, different types of cells can also be separated based on the light emitted by fluorescent molecules. This fluorescence may be due to naturally fluorescing materials inside a cell, or fluorescence-tagged antibodies. For example, fluorochrome is used to stain a protein of interest so that incident laser light of the appropriate wavelength allows the cells containing this protein to be detected.
As a cell passes through the laser beam, a pulse of photon emission is created. This pulse is detected by the photomultiplier tube and converted to a voltage pulse, which can be interpreted by the flow cytometer. The higher the intensity of fluorescence, the higher the voltage pulse.
Data interpretation
Each cell that passes through the laser light is detected as a separate event. Also, different types of detected light: forward-scatter, side-scatter, and specific wavelengths of fluorescence emission, is assigned a distinct channel. The data for each of these events is plotted independently and can be represented by two methods: histograms and dot-plots.
Histograms compare a single parameter, where intensity is plotted on one axis and the number of events is plotted on a separate axis. Dot-plots can compare more than one parameter simultaneously, where each event is displayed as a single point and the intensity values of two or three channels are represented on the various axis.
In this scenario, events that have similar intensities cluster together in the dot plot. While dot-plots can compare multiple parameters together, histograms are easier to read and understand. In many cases, dot-plots and histograms are not mutually exclusive, and in many flow cytometry experiments both types of graphs are plotted to represent and assess multi-parametric data.
Gating in flow cytometry
Gating is a method used in flow cytometry where more information is preferentially collected about a certain sub-population of cells. This adds further resolution to flow cytometry and can be used to analyze multiple parameters.
In a gated flow cytometry experiment, data is collected about one or more channels from the dot-plot. Then, a gate box is drawn and a subpopulation of cells are selected for more analysis. This subpopulation is highlighted in other plots that display information from alternate channels. This method helps to provide greater flexibility and single-cell resolution for each channel.
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