Flow Cytometry: Basics and Applications

Flow cytometer is a sophisticated and analytical tool which analyzes the phenotypic and functional characteristics of a cell in much lesser time. It measures the light scattering features, mainly forward scatter and side scatter properties of the cell. Optics, fluidics, and electronics are the main elements flow cytometer. Hydrodynamic focusing of the sheath fluid present in a sheath chamber enables the streamline motion of cells. This allows the encounter of each cell separately with the laser. A photodiode, photomultiplier tubes, optical filters, and beam splitters collect and emit the light or fluorescence in the form of signals. The signals are further processed by the electronic component and with the help of software, data are analyzed. For a cell to emit fluorescence, a number of fluorochromes (FITC, Alexa Fluor 488, Alexa Fluor 647 etc.) and dyes (propidium iodide, Hoechst, ethidium bromide etc.) are available. These fluorochromes may be conjugated with antibodies and emit fluorescence when excited by the laser. A diverse range of applications are possible using flow cytometer like apoptosis assay, cell cycle analysis, free radical generation assay, cytokine estimation, immunophenotyping, phagocytosis assay and many more. An advanced application of flow cytometer receiving attention is cell sorting. Individual cells or cell types can be sorted from a mixture of cells using physical fluorescence emission features of the cells named fluorescence assisted cell sorter.

Keywords: Apoptosis, Cell cycle, Cells sorting, Fluorochromes, Fluorescence, Immunophenotyping, Laser, Light scattering, Photomultiplier tube.