Cell sorting is the process through which a particular cell type is separated from others contained in a sample on the basis of its physical or biological properties, such as size, morphological parameters, viability and both extracellular and intracellular protein expression. The homogeneous cell population obtained after sorting can be used for a variety of applications including research, diagnosis, and therapy.

Methods of cell sorting fall into two major categories: fluorescence-activated cell sorting (FACS) and immunomagnetic cell sorting. Due to many years of refinement and increased demand for cell separation however, researchers are working to develop microfluidic sorting devices that have many benefits in comparison to the main types of fluorescence-activated cell sorting and immunomagnetic cell sorting methods.

Fluorescence-Activated Cell Sorting is also known as flow cytometry cell sorting, or by the acronym FACS, which is a trademark of Becton Dickinson and Company. Fluorescence-activated cell sorting utilizes flow cytometry to separate cells based on morphological parameters and the expression of multiple extracellular and intracellular proteins. This method allows multiparameter cell sorting and involves encapsulating cells into small liquid droplets which are selectively given electric charges and sorted by an external electric field. Fluorescence-activated cell sorting has several systems that work together to achieve successful sorting of events of interest. These include fluidic, optical, and electrostatic systems. The fluidic system has to establish a precisely timed break-off from the liquid stream in small uniform droplets, so that droplets containing individual cells can then be deflected electrostatically Based on the invention of Richard Sweet, droplet formation of the liquid jet of a cell sorter is stabilized by vibrations of an ultrasonic transducer at the exit of the nozzle orifice. The disturbances grow exponentially and lead to breakup of the jet in droplets with precise timing. A cell of interest that should be sorted is measured at the sensing zone and moves down the stream to the breakoff point. During the separation of the droplet with the cell in it from the intact liquid jet, a voltage pulse is given to the liquid jet so that droplets containing the cells of interest can be deflected in an electric field between two deflection plates for sorting. The droplets are then caught by collection tubes or vessels placed below the deflection plates. Flow cytometry cell-sorting yields very high specificity according to one or several surface markers, but one limitation is constituted by the number of cells that can be processed during a work day. For this reason pre-enrichment of the population of interest by immunomagnetic cell sorting is often considered, especially when the target cells are comparatively rare and a large batch of cells must be processed. Moreover, flow cytometry cell-sorters are complex instruments that are generally used only by well-trained staff in flow cytometry facilities or well-equipped laboratories and, since they are normally large in size, it is not always possible to place them inside a biological safety cabinet. Therefore, it is not always possible to ensure sample sterility and, since the fluidic systems can be cleaned but are not single-use, there is the possibility of cross-contamination among samples. Another aspect to be considered is that droplet-generation inside the instrument could lead to aerosol formations that are hazardous to the operator when using infectious samples. These last considerations are of particular importance when cell sorting is used for clinical applications -- for example cell therapy -- and should be performed under Good Manufacturing Practice (GMP) conditions. Researchers can use a variety of fluorescent dyes to design multi-color panels to achieve successful, simultaneous sorting of multiple, precisely defined cell types. Diagram A shows fluorescence-activated cell sorting of negative cell selection (undesired group) and diagram B shows FACS of positive cell selection (desired group).

Fluorescent dyes can act very differently. Generally, a fluorescent dye will be excited by a light source (a laser) at a particular wavelength and emit light at a lower energy and longer wavelength. The most common dyes act by binding to antigens presented on cells. Common antigens targeted are clusters of differentiation (CDs). These are specific to a certain type of cell. If you can identify which CD is presented on your cells of interest, then you can stain your sample with a fluorescent dye specific to it, and use fluorescence-activated cell sorting to separate the population of interest. However, there are many other mechanisms by which fluorescent dyes can act.

Some dyes are able to diffuse across membranes. By taking advantage of this property of the dye, users can characterize intracellular activity as well as surface-expression of proteins. For example, in dead cells, propidium iodide (PI) can penetrate the nucleus where it binds to DNA. The fluorescent signal of PI can be used to quantify DNA content for cell cycle analysis or to identify dead cells in a sample.

Certain fluorescent dyes can be used to characterize kinetic intracellular activity rather than fixing cells in formaldehyde and losing viable cells. The table below outlines dyes that can be used to measure several parameters of cytotoxicity caused by oxidative stress.

This experimental setup is just one example of the capability of flow cytometry. In FACS systems, these characterized cells can then be sorted and purified for further experiments.

MACS