Fluorescence-Activated Cell Sorting

Fluorescence-activated cell sorting (FACS) is a specific type of flow cytometric method. The use of this method has in the past decades increased exponentially in clinical laboratories and research laboratories. The FACS machine is a laser-based biophysical technology that measures multiple characteristics of individual cells in a stream of fluid. It can obtain both qualitative and quantitative information. The method is widely used in clinics and laboratories for cell counting, cell sorting, and biomarker detection. The modern FACS machine can be used for simultaneous detection of up to 20 different parameters and can efficiently separate the rare positive event from a large population.

The machine offers rapid detection of multiple characteristics of single cells. Very often, measured characteristics are cell size, DNA content, expression of cell surface proteins, cytoplasmic complexity, and intracellular proteins. Analysis is partially based on the light-scattering properties of cells and emission properties of the fluorescent markers used. The forward scattering and side scattering of light are used to measure cell size and complexity (granularity), respectively. The forward scattering is directly proportional to the cell size, where more forward scattering represents apparently larger cells. Higher side scattering means higher complexity, such as dividing cells or apoptotic cells. Apart from light-scattering properties, the FACS machine can read different fluorescent markers and can define cell populations according to the markers. Multiple fluorescent markers can be used, depending on the purpose of the analysis. In many analyses, two or more markers are being used. The fluorescent markers such as Alexa Fluor, phycoerythrin, and fluorescein isothiocyanate (FITC) are commonly conjugated to specific primary or secondary antibodies. Some specific dyes such as Propidium Iodide (PI), 7-Aminoactinomycin-D (7-AAD), and Hoechst display specific characteristics and are directly used as markers. Fluorescent markers are mainly dependent on the users’ experimental questions. For example, expression of cell surface proteins can be determined by antibodies against certain proteins (CD34, KIT, FLT3, etc.) to define stem cell characteristics. The DNA content of cells can be measured by DNA-binding dyes such as PI, 7-AAD, or Hoechst staining. Thus, depending on the specific biological questions, cells can be probed with specific fluorescent markers and then individual cell population can be counted or sorted by FACS.

In a FACS machine, cells pass through a tiny so-called flow cell with a stream created by a surrounding sheath of isotonic fluid. In the flow cell, cells pass through the laser beams. The flow cell contains one or more lasers for excitation of the fluorescent markers and several filters to separate emission bands. The most commonly used excitation lasers are violet laser (405 nanometers [nm]), blue laser (488 nm), and red laser (640 nm). Cells absorb characteristic wavelength bands depending on the fluorescent markers and then emit light of distinct wavelengths that pass through a series of filters. The emitted light signals are then detected by photomultiplier tubes and digitized for computer analysis. After cells pass through the laser field, the machine can analyze user-defined characteristics of the cell and can determine whether it falls within the defined parameters set for sorting. Then it uses an electrical field to separate and collect cells in three different tubes, such as positive, negative, and neutral. Modern FACS machines can read around 10 thousands of events per second and can efficiently distinguish an event in more than 10 thousands events. So it is possible to separate vary rare positive events in a large population of cells.

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