As well as being employed to qualitatively and quantitatively assess cell phenotype and function, flow cytometry can be used to separate particles or cells based on fluorescence labelling. Following isolation, these cells can then be used for further functional, molecular or biochemical studies. To allow sorting, a special flow cytometer is needed that is slightly more com plex than one required for normal flow cytometric analysis. However, this method will allow cells expressing different markers (and therefore bound by different fluorescently conjugated antibodies) to be separated from one another.
A key benefit of fluorescence-activated cell sorting (FACS), over other cell-sorting methods such as panning and bead enrichment, is that cells can be sorted to a high degree of purity. Populations can be isolated up to 95–98% purity, and there are ample downstream applications of such a pure population of cells, both experimentally and clinically. Cells isolated and purified via FACS can be expanded in vitro, transferred in vivo, used in cell assays or subjected to RNA or DNA analysis.
The general principles of FACS are the same as those for analytical flow cytometry in that cells are assessed based on fluorescence characteristics of the mixed cell population, either by binding of fluorescently conjugated antibodies or genetic encoding/ expression of a fluorescent protein. Cells are then gated to identify the cell population of interest to be sorted. For separation, the cell suspension is forced into a single stream of cells, but broken into droplets in such a way that each fluid droplet is likely to only contain one cell. The basic principle of this droplet generation is the same as that used in ink-jet printing technology. Fluorescence characteristics of each droplet, and therefore cell, are then measured. If the observed parameters match the required characteristics defined by the gating strategy, a charge will be applied to the droplet. The droplet then flows past an electrostatic deflection system that allows droplets to be deflected, or guided, into specific collection tubes based on their electrostatic charge (Figure1).
Fig1. Basic principle of electrostatic cell sorting. A stream of droplets is generated and then, by means of electrostatic deflection, either sorted into specific compartments or moved into the waste flow, depending on their surface marker expression.
At present, commercially available research-grade FACS instruments are able to sort and collect up to six separate populations at one time. However, to successfully separate cell populations, good markers are required in order to fluorescently mark populations of interest. Obviously, the brighter the marker the easier it is to separate the population of interest. The success of the sorting also depends upon the machine being appropriately set up and calibrated. Most frequently, FACS will be performed by a trained worker who is familiar with the instrument and the methodological back ground. This requires specialist training, often provided by the manufacturer of the instrument.
