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Flow Cytometry

At flow cytometry, you will find background information and articles on the flow cytometry method, detailed protocols, bioinformatic analysis tools and a flow cytometry forum for all your cell biology research questions and discussions.

Flow Cytometry Background

Flow cytometry (also called FCM) is a molecular biology method which is commonly used to count, quickly examine, analyze and sort cells or even microscopic particles suspended in fluid. Flow cytometers allow a simultaneous multiparametric analysis of the physical and/or chemical characteristics of single cells or particles flowing past an optical and/or a electronic detection apparatus, in a fluid stream using a beam of laser light.

The term cytometry derives from the Greek words for measurement (metry), and cell (cyto) giving "flow cytometry" the measurement of single cells as they flow past a detector.

Flow or cell sorting is a further functional capability of flow cytometry allowing the separation of types of cells through the use of electrical or mechanical forces to collect populations of cells with one or more physical or chemical characteristic set by the user.

Flow Cytometry Applications

Flow cytometry is commonly used in the analysis of the cell cycle, detection and analysis of apoptosis or necrosis, cell sorting, cell surface antigen detection for immunology, and stem cell analysis.

Flow Cytometry Measurements

Flow Cytomers can be used to measure the following parameters, depending on the machine, fluorophore and other:

• volume of cells
• morphological characteristics of cells
• cell surface antigens (Cluster of differentiation (CD) markers)
• detection of cell pigments (i.e chlorophyll or phycoerythrin)
• apoptosis (quantification, measurement of DNA degradation, mitochondrial membrane potential, permeability changes, caspase activity)
• cell viability assays
• assess DNA (e.g in cell cycle analysis, cell kinetics, proliferation)
• assess RNA
• chromosomal analysis and sorting (for DNA library construction or chromosome painting)
• assess for protein expression and localization
• transgenic products in vivo, particularly the Green fluorescent protein or related fluorescent proteins
• enzymatic activity
• and many other parameters

How Flow Cytometers Work?

A beam of light (usually laser light) of a single wavelength is directed onto a hydro-dynamically focused stream of fluid. A number of detectors are aimed at the point where the stream passes through the light beam; one in line with the light beam (Forward Scatter or FSC) and several perpendicular to it (Side Scatter (SSC) and one or more fluorescent detectors). Each suspended particle passing through the beam scatters the light in some way, and fluorescent chemicals found in the particle or attached to the particle may be excited into emitting light at a lower frequency than the light source. This combination of scattered and fluorescent light is picked up by the detectors, and by analysing fluctuations in brightness at each detector (one for each fluorescent emission peak) it is then possible to extrapolate various types of information about the physical and chemical structure of each individual particle. FSC correlates with the cell volume and SSC depends on the inner complexity of the particle (i.e. shape of the nucleus, the amount and type of cytoplasmic granules or the membrane roughness). Some flow cytometers on the market have eliminated the need for fluorescence and use only light scatter for measurement. Other flow cytometers form images of each cell's fluorescence, scattered light, and transmitted light.