The acronym FACS (Fluorescence Activated Cell Sorting) and flow cytometry are used interchangeably. FACS is a powerful method used to study and purify cells. FACS has a wide application in immunology and cell biology and other fields of biology.
Individual cells held in a thin stream of fluid are passed through one or more laser beams cause light to scatter and fluorescent dyes to emit light at various frequencies. Photomultiplier tubes (PMT) convert light to electrical signals and cell data is collected. Cell sub-populations are identified and sorted at high purity (~100%). FACS instruments generate three types of data:
| Forward Scatter(FSc) | Approximate cell size | |
| Side or Orthogonal Scatter(SSc) | Cell complexity or granularity | |
| Fluorescent Labeling | Used to investigate cell structure and function. | |
Forward and side scatter are used for preliminary identification of cells. In a peripheral blood sample, lymphocyte, monocyte and granulocyte populations can be defined on the basis of forward and side scatter. Forward and side scatter are used to exclude debris and dead cells.
Fluorescent labeling allows investigation of cell structure and function. Cell autofluorescence is generated by labeling cell structures with fluorescent dyes. FACS collect fluorescence signals in one to several channels corresponding to different laser excitation and fluorescence emission wavelength. Immunofluorescence, the most widely used application, involves the staining of cells with antibodies conjugated to fluorescent dyes such as fluorescein and phycoerythrin. This method is often used to label molecules on the cell surface, but antibodies can be directed at targets in cytoplasm.
In direct immunofluorescence an antibody to a molecule is directly conjugated to a fluorescent dye (such as lymphocyte surface marker CD4). Cells are stained in one step. Conjugation of Monoclonal Antibodies provides a thorough discussion of antibody conjugation.
In indirect immunofluorescence the primary antibody is not labeled. A second fluorescently conjugated antibody is added which is specific for the first antibody. For example, if the anti-CD4 antibody was a mouse IgG then the second antibody could bea rat antibody raised against mouse IgG. Immunofluorescence examples:
Alcohol run through sample line removes residue from samples containing contamination and pathogens from previous samples. This can be followed with a propidium iodide solution to label dead material for removal from subsequent data.
FACS can be used to investigate cell biology. Calcium flux can be measured using Indo-1 markers. This can be combined with immunofluorescent stain. For example, identify T cell subpopulations by immunofluorescence and measure calcium flux in response to an activating signal. Rhodamine-123 stains mitochondrial membranes is used to measure cellular activation. Rhodamine-123 is rapidly pumped out of some cells (for example hematopoietic stem cells).
CFSE binds to cell membranes and equally distributes when cells divide. Cell divisions in a period of time can then be counted. For example, labeling a population of cells with CFSE in vitro and reintroduce them in vivo. After a few days, the cells could be sampled and the amount of division measured. For example: Metabolic characteristics such as calcium flux, mitochondrial activity, pH, and free radical production can be measured in live cell populations in real time.
FACS is used to measure gene expression in cells transfected with recombinant DNA. This is achieved directly by labeling the protein product, or indirectly by using a reporter gene in the construct. Direct immunofluorescent labeling allows quantification of the product, and is suitable for relatively abundant proteins expressed on the cell surface. Indirect detection by reporter genes allows detection of transfectants at lower levels which cannot be detected easily by immunofluorescence. Examples of reporter genes are beta galactosidase and Green Fluorescent Protein (GFP). Beta galactosidase activity can be detected by FACS using fluorogenic substrates such as fluorescein digalactoside (FDG). FDG is introduced into cells by hypotonic shock and is cleaved by the enzyme to generate a fluorescent product trapped within the cell. One enzyme can generate a large amount of fluorescent product.
Cells expressing GFP constructs will fluoresce without addition of a substrate. GPF mutants are available which have different excitation frequencies, but emit fluorescence in the same channel. In a two-laser FACS, it is possible to distinguish cells which are excited by different lasers and assay two transfections at the same time.
Fluorescent dyes allow analyses of simultaneous parameters that can refine cell subpopulations. The fluorescent dyes that you can use will depend upon which Facility instrument you use.
When different fluorescent dyes are used, signal spillover can occur between fluorescence channels. This needs to be corrected by setting compensation. Theory and Practice of Compensation provides a thorough discussion of compensation. Setting correct compensation is important for obtaining accurate results when using multiple colors.
If FACS could be of value for your experiment, contact Facility staff for advise or discuss your proposed experiment with other users with similar interests. You are welcome to explore the Shared FACS Facility lending library in Beckman B015.
Copyright©1998 Stanford University(rev 6/19/2003)