Flow Cytometry Shared Service

Director: Ferenc Livak, M.D.

Flow cytometry is a powerful technique tailor made for making measurements on single cells. Thus it is tailor made for examining cancer cell lines as well as evaluation of clinical response to therapeutic agents used to treat hematologic malignancies. Solid tissues and adherent cells that grow in culture can also be measured after first disaggregating the individual cells by mechanical or chemical means. Size, shape, granularity, protein content, DNA content, intracellular pH and calcium concentration are the most common cellular parameters that can be measured by the flow cytometer. The cells may be examined live or fixed.

The term flow cytometer is often used synonymously with fluorescence activated cell sorter (FACS). Cells are stained with a variety of different dyes (fluorochromes) that bind to proteins or nucleic acids, or with dyes whose fluorescence emission spectrum usually change with changes in calcium ion hydrogen ion concentration. In order to identify specific proteins, fluorescent dyes like fluorescein (green emission) or phycoerythrin (orange emission) are covalently attached to monoclonal antibodies that will only bind to those cells that are expressing the protein to which the antibodies were raised.

DNA dyes like propidium iodide allow the researcher to identify which cells are diploid (G0, resting, non-dividing cells, or any cycling cells in G1), which cells are in S phase of the cell cycle during which DNA is replicated, and which cells are G2 or in mitosis. Tumor cells are often aneuploid, they have too many or too few chromosomes compared to the normal diploid DNA number for the species.

Once labeled, the cells are made to flow rapidly through the flow cell, or stream in air, where they are illuminated by a focused laser beam at a single wavelength (488 nm wavelength light from an argon laser). The fluorochromes must be matched to absorb the light emitted by the argon laser. As each cell intercepts the laser beam it scatters some of the illuminating blue light. This acts as a trigger signal to alert the computer to begin making measurements. The intensity of the scattered laser light tells the operator about the diameter, shape, and granularity of the cell. The cell may also fluoresce in the green or red region of the visible spectrum depending on the dyes that were applied to the cell.

Light of different wavelengths simultaneously emitted from a single cell can be separated by optical elements like glass filters and dichoic beam splitters. Light in different regions of the spectrum is routed to different photodetectors and can be measured simultaneously in addition to the scattering information. The intensity of the fluorescence can be used to quantitate the amount of DNA, protein or other molecules being measured by comparing the signal to known standards. Typically several thousand cells are acquired per tube and statistics for the entire population are generated at the end of the run.

Some flow cytometers have the option for “sorting”, which by making the appropriate measurements, the computer makes the decision to sort or isolate a single cell by applying a charge to the cell just as it leaves the flow cell. The cell is electrostatically deflected into a collection tube. Any cell can be sterilely sorted and recovered alive based on any combination of light scatter and fluorescence. The cell type of interest can be separated from a complex mixture of cell types even though it may be an extremely rare or minor subpopulation.

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