Fluorescence-activated cell sorting

Fluorescence-activated cell sorting (FACS) is a method of analyzing and physically separating cells based on their fluorescence-labeled surface or intracellular features. FACS is a specialized form of flow cytometry in which cells are automatically characterized according to detected fluorescence signals: each cell successively passes through a laser beam, and the emitted fluorescent light is measured and interpreted. Based on their fluorescence profile, the cells are then sorted electrostatically into separate containers. This technique allows for the precise isolation of specific cell populations from heterogeneous samples for further functional or molecular analysis.

How fluorescence-activated cell sorting (FACS) works

In flow cytometry, cells are labeled with a fluorescent tag that targets the desired property. During the fluorescence-activated cell sorting process, these tagged cells pass individually through a laser beam in a liquid flow. The laser excites the fluorescent tag, and the instrument’s detectors record and analyze the emitted fluorescence signal. Based on their fluorescence profile, the cells are then sorted electrostatically into separate containers.

Key components of a fluorescence-activated cell sorting instrument

A FACS instrument consists of three main components: the fluidic system, the optics system, and the electronics system. The fluidic system ensures that cells flow individually and uniformly through the measuring chamber. The optics system uses lasers and detectors to capture fluorescence and scattered light signals. The electronics system processes these signals in real time and controls cell sorting. Together, these components enable the precise analysis and separation of complex cell mixtures.

Applications of fluorescence-activated cell sorting in research & medicine

Immunology & cell biology

Fluorescence-activated cell sorting is a key tool for identifying and isolating specific immune cell types, such as T cells, B cells, natural killer (NK) cells or dendritic cells. These cell types can be precisely filtered and separated from heterogeneous samples using fluorescence-labeled antibodies that selectively bind to their surface markers. Therefore, FACS is a valuable technique for immunological research and diagnostics.

Generally, FACS plays an important role in investigating cell surface markers of various cell types, such as immune cells, cancer cells or stem cells. This method enables researchers to identify, quantify and isolate cells based on their unique surface protein profiles.

Cancer research

In cancer research, fluorescence-activated cell sorting is used to detect and specifically enrich tumor cells based on surface or intracellular markers. This technique allows for a thorough examination of cancer cell populations in blood, tissue, or cell cultures, even when they are present in small quantities. DNA dyes, such as propidium iodide, and apoptosis markers can be used to determine cell cycle status and cell viability in order to evaluate responses to cancer treatment or resistance mechanisms by FACS.

Stem cell research

Fluorescence-activated cell sorting allows for the targeted isolation of stem cells from complex cell mixtures, such as blood or tissue samples. Characteristic surface markers are used to select either pluripotent or differentiated cell types. This allows for the isolation of pure stem cell fractions for research and therapeutic applications.

Microbiology

FACS has also gained importance in microbiology. Microorganisms are labeled with either nonspecific DNA dyes (e.g. SYBR Green) or specific antibodies to sort them according to their fluorescence profiles. This allows, for example, the targeted enrichment of pathogenic bacteria or the investigation of the physiological states of microbial subpopulations. FACS also supports the analysis of bacterial biofilms and of virus-infected cell populations.

Advantages of using fluorescence-activated cell sorting

High precision & purity

Fluorescence-activated cell sorting provides exceptionally high sorting accuracy because it classifies cells based on sensitive, clearly defined, high-resolution fluorescence signals. Simultaneously evaluating multiple markers enables the precise identification and isolation of rare cell types with over 95% purity. This is particularly important for subsequent applications, such as RNA sequencing, functional cell testing, and clonal expansion.

Multiparametric analysis

A key advantage of fluorescence-activated cell sorting is the ability to analyze numerous parameters per cell simultaneously, such as surface proteins, intracellular signals, DNA content, and metabolic activity. Depending on the instrument configuration, 10 or more fluorescence channels can be analyzed at once, providing a detailed cellular profile.

Speed & efficiency

Modern FACS instruments can analyze and sort several thousand to over 50,000 cells per second. This high throughput rate enables the processing of large sample volumes in a short amount of time, a significant advantage for clinical studies and high-throughput screening. Thanks to high-speed processing and sorting, even rare cellular events within a cell population can be reliably captured.