Flow Cytometry is a means of identifying and measuring certain physical and chemical characteristics of cells or particles as they travel in suspension. The benefit of flow cytometry is the rapid simultaneous measurement of several parameters on a cell by cell basis. Flow cytometry uses fluorescent probes to identify and characterize cells or particles. Cells or particles tagged with fluorescent molecules enter the cytometer via a fluid stream. The cells then pass by a laser, which emits a specific wavelength of light. The fluorescent probes are excited by the laser and then emit light. The fluorescent signal is detected and amplified, then translated into an electronic signal, which is sent to the computer. Information about the size and granularity of a cell is recorded, as well. The result is a visual presentation describing an individual or group of cellular events. The cells or particles can be separated by sorting, or the information can be collected and analyzed.
There are a number of applications for flow cytometry, including, but not limited to:
- Immunophenotyping- Using fluorescence-conjugated antibodies directed toward a protein(s) of interest, cells expressing that protein(s) on the surface or intracellularly may be detected by flow cytometry.Specific cell types may be distinguished within a mixed population using multiple fluorescence-conjugated antibodies.
- Transfection efficiency may be determined when a fluorescent protein (i.e. GFP) is used as a marker.
- Apoptosis measurement- Several flow cytometric methods to detect apoptosis are available. Cells can be stained with Annexin V or 7ADD.
- Cell cycle analysis- Fixed cells are stained with a dye that binds to DNA (e.g. propidium iodide,ethidium bromide,DAPI).
- Fluorescent intensity is used to determine the amount of cellular DNA present in each cell (i.e. two copies of a genome have roughly twice the fluorescent intensity of one copy).
- Cell proliferation- Carboxyfluorescein diacetate, succinimidyl ester (CFSE) is a dye that diffuses into cells and is passed from parent to daughter cells so that the cells of each generation have half the fluorescent intensity of their parent cells.
- Cell sorting- A particular subset(s) of cells may be sorted from a mixture of cells based upon particular properties.
- Membrane potential- Bacterial membrane potential may be analyzed using DiOC2, which exhibits green fluorescence in all bacterial cells, but shifts to red fluorescence as the dye becomes more concentrated in cells with larger membrane potentials.Mitochondrial membrane potential may be analyzed in the same manner with JC-1.
- Live/dead bacteria discrimination- You can test how fast an antibiotic is killing microbes: live cells have intact membranes and are impermeable to dyes such as propidium iodide, which only leaks into cells with compromised membranes. Thiazole orange enters all cells, live and dead, to varying degrees. Thus a combination of these two dyes provides a rapid and reliable method for discriminating live and dead bacteria.