Flow cytometry is one of the essential techniques for cell analysis. This article describes the basic mechanics of flow cytometry, measurement devices using flow cytometry, how flow cytometers work, and how they can be used.
Flow Cytometry is a technique for cell analysis that was introduced in the 1950s. Fluid-containing cells flow at high speed through a capillary with an observation window. The cell then passes in front of the observation window, and this single cell is observed.
Flow cytometry can measure cell characteristics such as cell size, cell count, and cell cycle. This provides information on individual cells from a heterogeneous cell population.
A flow cytometer is a cell analysis device that uses flow cytometry technology. Among flow cytometers, those with the ability to separate cells are called cell sorting, cell sorters, or FACS (Fluorescence Activated Cell Sorter).
In recent years, cell sorters have increasingly been used in a similar sense to flow cytometers. The cell sorter is a device that separates and collects the cells analyzed by flow cytometry. This classification and collection is called sorting.
Flow cytometers consist of two major components. The first is a fluid system for flowing and handling cells. The other is an optical system that includes a light source, a signal detector, and a processor for data acquisition.
In a fluid system, cells are arranged one by one in a row in a buffer called a sheath fluid by the flow of the fluid. In the optical system, a laser beam is shone on a row of cells, and the scattered light and fluorescence are measured by Photomultiplier Tubes (PMTs). This is the mechanism by which information on each cell can be obtained.
The cell sorter charges the target cells with this information. The cells are then separated by a high-voltage polarizer.
Because cells are measured in an undisturbed flow (laminar flow), it is possible to measure cells without damaging them.
Flow cytometers are used for cell cycle evaluation based on DNA content, as well as cell surface marker analysis using fluorescently labeled antibodies and intracellular introduction analysis of fluorescently labeled macromolecules. It is also used for sorting specific cells.
When conducting cell biology research experiments using flow cytometry, the first step is to plan what color fluorescent dye to assign to each antibody.
To prevent color mixing during analysis and to avoid confusion as to which cells are which, dyes are assigned while taking into consideration the maximum excitation wavelength, maximum emission wavelength, and luminance, as well as the type of laser used in the flow cytometer.
Next, follow the steps in the cell staining protocol. Cell staining can be done by direct staining, indirect staining, or intracellular staining. The staining method is selected according to the type of cell to be measured and the item to be measured.
After fluorescent dye assignment and staining, the specimen is filtered to remove cell clumps and strands (aggregates). This is because flocculence clogs the flow path and interferes with measurement.
After filtration, perform flow cytometry and analyze the results.
Flow cytometers are used in the study of cell biology as well as molecular biology. In particular, fluorescent molecules are used to analyze the function of cell surfaces and inside cells and make cell functions visible.
Flow cytometers are used to detect specific cells for molecular beacons and protein fluorescent labeling when conducting molecular biology studies. This measurement makes it possible to examine the immune system, etc.
Compared to fluorescence microscopy, analysis using a flow cytometer has the following features: "multiple parameters can be analyzed simultaneously," "quantitative analysis is possible," and "analysis speed is fast.