Fluorescence-Activated Cell Sorting (FACS)

What is FACS?

FACS (Fluorescence-Activated Cell Sorting) is a specialized type of flow cytometry that sorts a heterogeneous mixture of cells based upon the specific light scattering and fluorescent characteristics of each cell. Cells are first tagged using fluorescent antibodies that bind to relevant proteins on target cells. Expression of fluorescent proteins or intercalating dyes can also be used to label cells before sorting. The mixture of suspended cells rapidly flows through the instrument's flow cell in a single file. Individual cells pass a laser beam, and a detector measures the fluorescence, forward-scattered light (FSC), and side-scattered light (SSC). FSC and SSC are used as an indication of the cell’s size and granularity. Fluorescence at several wavelengths can be detected simultaneously depending on the instrument’s specific lasers, filters, and detectors. Cells are then physically sorted into different containers based on detected measurements and user entered parameters.

For experiments where highly pure populations of cells need to be isolated, FACS provides a method that achieves high purity, viability, and throughput.

Fluorescence-activated cell sorting

A fluorescence-activated cell sorter provides the ability to separate cells identified based on size and/or fluorescence. Droplet based cell sorters first analyze the particles, but also have hardware that can generate droplets and a means of deflecting or directing droplets containing the desired particles into a collection tube. Droplets can be formed by using high-frequency (cycles/second, Hz) vibration of the nozzle at an optimal amplitude (in volts). This is typically created by a piezoelectric crystal. As cells pass through the laser interrogation point, light emitted from the cell is collected via detectors, and this signal is then processed by the instruments electronics which then tells the instrument to apply a charge to the droplet. The charged droplet can then be deflected into the appropriate tube for collection.

Other Cell Sorting Methods

There are several different methods for cell analysis; the oldest is the Coulter principle, which measures a change in impedance to determine particle volume and is still used for counting and sizing. Analytic techniques were later supplemented by preparative techniques such as magnetic separation using magnetically labeled antibodies to separate cells in bulk. Magnetic cell separation must be done using extracellular markers and typically can be done using positive selection or the depletion of unwanted cells. Using cell sorting based on flow cytometry enables cell identification and cell sorting at the individual cell level. Fluorescence-activated cell sorting, also known as fluorescence-assisted cell sorting, allows for several parameters to be used to identify the cells of interest, and single-cell sorts can be performed.