In a heterogenous population of cells, researchers are often interested in one or two subpopulations. Studies that, for example, examine the differentiation of cells following treatment with a specific growth factor, cytokine, or drug may wish to understand the differences between these two different cell states or what drives one state into another. FACS enables the identification and sorting of the cells based on their specific phenotype using fluorescent labels. These labels, typically fluorescent antibodies that recognize molecules on the cell surface, or fluorescent fusion proteins that are heterologously expressed in the cells, emit light when illuminated with light of a specific wavelength. These properties can then be used to sort the cells into more homogenous subpopulations that can be interrogated in future experiments. Kitti Dora Csalyi, head of the FACS facility, explains: “The power of this technology lies in its ability to analyze thousands of cells in a very short time frame. The data provided by flow cytometry offers an unbiased, comprehensive view of the cell population within your sample.” At the Perutz, the FACS facility supports more than two-thirds of the research groups by providing the necessary equipment and expertise to sort and analyze objects up to 750 µm, delivering consistent and high-quality data.
The facility houses two high-throughput cell sorters – including one spectral cell sorter –, one user-friendly cell sorter, three cell analyzers, and one large particle sorter. Cell sorters separate and isolate specific cell populations based on their characteristics, making them available for downstream analysis, while cell analyzers measure and analyze cell populations without physically sorting them. The spectral flow cell sorter, a cutting-edge technology, can simultaneously monitor over 40 different wavelengths, permitting high-resolution sorting of heterogeneous cell populations. The large particle sorter enables the sorting of 3D cell clusters and zebrafish larvae. “Due to their size, these particles are sorted based on an air pressure system, whereas the sorting in our other instruments is based on droplet charging,” Kitti explains.
Making Cell Sorting Accessible to All
Beyond routine maintenance of the machine park, the facility team, comprising Kitti Dora Csalyi and her colleagues Thomas Sauer and Endre Kiss, bring their extensive expertise in experimental design and sample handling to promote best practices. A primary goal of the facility is to offer hands-on training and make FACS accessible to every scientist in-house. Endre Kiss notes: “Part of my role involves training new users and providing technical assistance.” This ensures that cell sorting and FACS analyses can be performed by trained users outside of the normal facility hours of operation.
The FACS-tastic Technology
In FACS, cells are first labeled with fluorescent markers that bind to specific proteins, cellular components, or DNA. Kitti explains: “When the cell suspension is loaded into a cytometer, the cells are transported into a flow cell where the sample interrogation occurs.” As these cells pass through a laser beam within the FACS machine, they emit light at varying wavelengths, depending on the specific labels that they display. Detectors capture this fluorescence, providing real-time information on the phenotype of every cell. In cell sorters, electrical charges direct the cells into different collection tubes based on their fluorescence profiles, as well as physical characteristics such as cell size. This allows researchers to isolate specific cell types for further study, making FACS an indispensable tool in molecular biology.