Flow Cytometry Applications
Flow Cytometry allows researchers to analyze multiple characteristics of single cells simultaneously, in the form of a fluid stream flowing through a beam of light. Properties that can be measured include size, number, internal complexity, and granularity. These measurements can be used to define different types of cell within a heterogeneous population as well as the purity of isolated subpopulations. These measurements can also indicate the level of apoptosis or proliferation within a population.
Fluorescence from labeled antibodies or ligands can also be detected, to quantify the expression of cell surface or intracellular molecules.
Basic Staining Procedure
• Make a single-cell suspension for culture or tissue sample
• Incubate cells in tubes or microtiter plates
• Add unlabeled/fluorochrome-labeled antibodies
• Analyze on the flow cytometer
Cells can be sorted with the use of a specific type of flow cytometry known as FACS.
Measuring Size and Complexity
Sheath fluid forces the cell fluid through a nozzle which only allows one cell to pass through the light beam at a time. The cells create both forward scatter (used to measure size), and side scatter (used to measure internal complexity). These two readings can be used to identify the cell. For example, granulocytes create both a high forward scatter and side scatter, while lymphocytes produce low forward and side scatters.
At the same time, the light beam will excite any fluorochromes/fluorophores present in the cell; these stains mark specific proteins (including antibodies) of interest. Sensors known as photomultiplier tubes (PMTs), within the flow cytometer, detect the light emitted from these fluorophores.
Each sensor will detect fluorescence at a specific wavelength (therefore only a certain few fluorophores) with the help of an appropriate filter. Short pass filters only allow wavelengths below a certain value, while long pass filters allow wavelengths above a certain wavelength.
Fluorescence Activated Cell Sorting (FACS)
FACS can separate live cells into subpopulations depending on their fluorescent labeling, i.e. which fluorophore has been used to stain them. The technology uses deflection plates to attract or repel cells based on the charge of the fluorophore.