by Julia Riley
Fun Rating: 4/5
Difficulty Rating: 3/5
What is the general purpose?
Fluorescence-Activating Cell Sorting (FACS) is a type of flow cytometry that sorts a mixture of cells based on the physical and fluorescent characteristics of each cell. By separating out subsets of a heterogeneous population, FACS allows for understanding the characteristics of single cell populations without the influence of other cells.
Why do we use it?
FACS sorting is used to separate a heterogenous (mixed) population of cells into different groups. Depending on the lab and aims of the experiment, cells will be sorted based on different parameters, but a common example includes sorting cells by levels of gene expression. This is particularly powerful for CRISPR screens, which are large experiments where many changes are introduced into an organism’s DNA to find important or interesting genes/regulatory elements. In this technique, FACS can be used to sort cells based on gene expression in order to determine which disruptions to a gene cause the greatest increase or decrease in gene expression.
How does it work?
Fluorophores, fluorescent molecules, are attached to antibodies or dyes that bind to the cells. As these cells flow single file past a laser of a particular light wavelength, these fluorophores emit light, allowing the cells to be distinguished from one another based on fluorescent intensity.
Think of this process as taking a whole school population, say an elementary school of 500 students, and giving each student glowsticks representing their characteristics. You could give students one glowstick for each grade, so fifth graders would get five glowsticks. If you only wanted to divide the population by grade, you could stop here. However, if you wanted a more specific subset of the population, maybe divided by which sports they play, who their teacher is, or what their favorite class is, you could give the students glowsticks of different colors to represent each of these traits. However, when sorting students you would have to be careful with your measurements. If a first grader had a massive glowstick, it could be as bright as a fifth grader’s five glowsticks. This is where cell granularity comes in. The size and complexity of each glowstick is also important while sorting. Just like flow sorting, you are separating a larger population into smaller groups based on measured characteristics of your choosing.
Preparing Samples for FACS
To prepare samples for sorting with FACS, cells must be labeled with fluorophores. Depending on what you want to measure with FACS, there are lots of options for which fluorophores to use! ThermoFisher has a helpful website where you can explore fluorophores based on excitation wavelength and peak emission wavelength.
Generally, you will incubate your cells with the fluorophores so they can bind to the target markers on the cell surfaces. After incubating, cells are washed to remove any unbound fluorophores. Once washed, cells are resuspended, which means to disrupt the solution so the cells return to floating, to remove residual color, maintain cell viability (make sure the cells are alive and healthy) during the sorting process, and prepare them to be run through the flow cytometer.
Sorting the Cell Samples
The mixture of cells flows through the flow cytometer single file. As each cell passes the laser beam, the flow cytometer measures the fluorescence, forward-scattered light (FSC), and side-scattered light (SSC). FSC serves as a metric for cell size while SSC depicts the granularity (the complexity within a cell, like the number of internal organelles) of the cells. Think back to the glowstick analogy… the size and type of glowstick are very important to consider! Fluorescence indicates the presence and quantity of the bound fluorophores. Depending on what the fluorophores are bound to, fluorescence can serve as a measurement of gene/protein expression, quantity of particular surface markers, and even intracellular cytokines (proteins cells secrete to communicate with one another). Based on these measurements and the user’s parameters, cells within the sample are then physically separated into different containers. Researchers can either end here with the data from the flow cytometer or perform further testing, such as next generation sequencing, to better understand each population.
Figure 1: Process of fluorescent-activating cytometric sorting (FACS). Image generated by author using BioRender.
