METHODS AND TECHNOLOGIES

Flow cytometry, or fluorescence-activated cell sorting (FACS), is a technique used to count and analyze the physical and molecular characteristics of particles, such as cells or plastic beads, suspended in a liquid stream. A primary application of this method involves using fluorescent dye-labeled probes, such as antibodies, receptors, or streptavidin, to detect specific properties of individual cells or cell populations at the single cell level. FACS is a method for the quantitative analysis of surface markers and intracellular proteins based on the specificity of antigen-antibody interactions, carried out with fluorescent labeled antibodies.

In the FACS process, cells are guided past a bundled laser beam in a narrow stream by hydrodynamic focusing, aligning them in single file. A monochromatic laser beam of an appropriate wavelength excites the fluorescent dyes on the labeled antibodies, which causes electrons in the dye molecules to move to a higher energy state. After the laser pulse, as the electrons return to their original energy level, they emit energy in the form of photons which are detected by a photodetector. The intensity of the photon concentration (emitted fluorescence) is directly proportional to the quantity of bound antibodies on each cell. Additionally, light scattering and diffraction during the process provide information about cell size and structural features, such as the granularity of the cytoplasm and the size of the nucleus.

FACS can measure multiple parameters simultaneously by using different fluorescent dyes. These dyes can share the same excitation wavelength but have distinct emission spectra, enabling the differentiation of various signals specific to each dye.

The most important areas of application for FACS analysis include

●Lymphocyte typing (immune status)

●Immunophenotyping of non-Hodgkin’s lymphomas

●Flow cytometric crossmatch

●Checking the purity of cell suspensions