This section provides an overview for flow cytometers as well as their applications and principles. Also, please take a look at the list of 4 flow cytometer manufacturers and their company rankings.
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A flow cytometer is a device used to perform a measurement method called flow cytometry.
In this method, a laser beam is shone on cells in a fluidized liquid sample to detect scattered light and fluorescence emitted from the cells, and to measure characteristics such as cell size, number, and intracellular and surface antigens.
Compared to fluorescence microscopy, flow cytometer have advantages such as "simultaneous quantification of multiple measurement items" and "high analysis speed. On the other hand, flow cytometer is not suitable for observation of morphological characteristics of cells.
Flow cytometers are widely used for research in the biological and medical fields that handle cells, as well as for clinical testing and treatment. The types of cells that can be measured vary. Peripheral blood leukocytes and other cells are analyzed for the purpose of disease diagnosis, and cultured animal and plant cells, adult stem cells, tumor-initiating cells, and other rare cells are also analyzed for characterization.
Other cell types that can be measured include microorganisms, marine organisms such as plankton, sperm, yeast, and latex beads.
Flow cytometers consist of three main components: a flow path system, an optical system, and an electrical system. The flow path system has the function of taking in a sample and flowing it into a flow cell, and a laser beam is applied to the flow cell from the optical system, which consists of a light source, lens, filter, and a detector that generates a photocurrent.
The fluorescence emission from the cells is then detected and analyzed by the electrical system. The flow path system consists of tubes, valves, and pumps, and the fluorescently labeled cells suspended in the sample are aligned in a row by the flow path system for analysis.
When laser light passes through the cells, called the interrogation point, the light is scattered and at the same time, fluorescence is emitted by excitation of the fluorescent dye bound to the cells, and this scattered light and fluorescence are detected as a signal by the detector. There are two types of scattered light signals: forward scattered light (FS) and side scattered light (SS).
Different detectors are used to detect FS and SS, with FS reflecting cell size and SS reflecting intracellular structure. The detected signals are converted into data in an electronic system and finally can be interpreted by software.
Flow cytometers are divided into two types: cell analyzers and cell sorters. Cell analyzers are devices that analyze cells. Cells in the sample solution, which is wrapped in a sheath liquid, are aligned one by one by a flow cell and flow through the detection section. A laser is then irradiated and the cells are analyzed by detecting light scattering and fluorescence emission.
Cell analyzers are simple and easy to operate because the emphasis is on analysis. In addition to cell analysis, cell sorters can also fractionate cells of interest. In addition to analyzing the structure, size, and proportion of cells, the cell sorter can also examine the distribution of cells at high speed and preparative sorting.
Cells of interest are given a positive or negative charge to form droplets. Only the droplets of charged target cells are collected in a test tube or micro tube, by a polarizer, with a voltage applied to change their direction of movement. Cell sorters are more complex and require more skill than cell analyzers.
There are two types of antibodies used in flow cytometry for cell detection: polyclonal antibodies and monoclonal antibodies. Polyclonal antibodies are purified from sera collected after immunizing animals with antigens. These antibodies are mixtures of antibodies that recognize and bind to multiple epitopes (antibody binding sites).
To produce monoclonal antibodies, animals are immunized with antigens and then the antibody-containing B cells are fused with myeloma (cancer cells). The resulting hybridoma secretes antibodies that are monoclonal antibodies. This antibody recognizes only one epitope.
Most antibodies for flow cytometry are monoclonal antibodies because they improve the accuracy of the experiment and the specificity of the detection of the target substance (antigen).
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