Flow cytometry and cell sorting (FACS)

About this technique

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Flow cytometry and cell sorting

Flow cytometry allows rapid multiparametric analysis of fluorescently labeled cells and particles in suspension. The suspension is passed in front of a laser causing excitation of the attached fluorophores. The emitted light is then split into different wavelengths (colours) and collected using a number of photomultiplier tubes. High-speed analysis allows fast and accurate quantitation of population statistics and fluorescence intensity changes. As well as population analysis of the suspended cells or particles, cell sorters allow cells to separated and isolated on the basis of the attached fluorophores. These techniques can analyse and sort both living and fixed cells.

Flow cytometry is routinely used in clinical diagnostics, especially leukaemia and AIDS, but has many applications in research. These include:

• immunophenotyping, where cells are labeled with fluorescently tagged antibodies
• DNA and RNA analysis
• cell viability
• cytokine expression
• kinetics and proliferation
• intracellular calcium
• oxidative burst
• intracellular pH
• phagocytic activity
• mitochondrial membrane potential
• identification of phytoplankton populations

Flow cytometry analysisgenerates bivariate dot-plots and histograms that can be used to calculate population statistics and changes in mean fluorescence intensity within the sample. Further information on these techniques is listed below, but this list is by no means exhaustive.

Immunophenotyping

Cells can be rapidly identified and quantified by staining with fluorescently tagged antibodies to both extracellular and intracellular antigens. A large number of fluorophores are available and, depending on the instrument, up to eight different colours can be run simultaneously. High-speed instruments can run up to 10,000 cells per second allowing identification of rare populations.

DNA analysis and ploidy determination

Using fluorescent dyes that bind stoichiometrically to DNA (fluorescence is directly proportional to DNA content) researchers can perform a number of assays including measurement of cell cycle, cell kinetics and proliferation. A further application is determining ploidy (the number of chromosomes in a cell), which is particularly useful in plant breeding programs.

Cell viability and apoptosis

Flow cytometry offers numerous assays for the study of apoptosis and cell death. These include DNA degradation, mitochondrial membrane potential, staining with antibodies specific for caspase proteins, and the commonly used Annexin V, 7AAD kits for detection of early apoptosis and cell death. Viability markers such as Propidium Iodide (PI) or 7AAD are only able to enter cells with a compromised membrane and are commonly included in flow cytometry assays to ensure only viable cells are analysed.

Cell sorting

Fluorescence activated cell sorting (FACS) allows the simultaneous separation of up to four pure populations from a heterogeneous suspension sample of cells, nuclei, bacteria or phytoplankton. Particles varying from 200 nm to 60 µm in size, can be sorted at a speed of up to 90,000 cells per second. The collected cells or particles may then be used for subsequent analysis, tissue culture or injection into experimental animal models.

Aquatic pico phytoplankton

Rapid and accurate identification of vast numbers of phytoplankton cells can be achieved by taking advantage of inherent autofluorescent properties of different phytoplankton species. Detection of cell pigments such as chlorophyll and phycoerythrin together with side scatter populations can provide “fingerprints” for different phytoplankton cells and therefore allow researchers to quantify these populations in marine and freshwater samples.

Soluble protein quantitation

Bead-based assays allow detection and quantification ofoligonucleotidesand soluble proteins from serum, cell supernatant and tissue homogenates. The benefits offered over traditional ELISA assays include much lower sample volume, analysis of multiple analytes in the same sample (multiplexing) and a higher dynamic range of sensitivity. Applications include the detection of cytokines (replacing conventional ELISA), nucleic acid assays, serology isotyping and enzyme-ligand research. Samples are run in 96-well plates with sample volumes as low as 25 µl capable of multiplexing of up to 100 analytes per well. Software is able to create standard curves using a number of fitting methods and can automatically calculate analyte concentrations.