The FACS process begins by labeling cells with fluorescent antibodies or dyes that bind to specific proteins or nucleic acids in the cells. These fluorescent markers allow the flow cytometer to detect and distinguish between different cell types based on their expression of target molecules. Fluorescent proteins and intercalating dyes can also be used to label cells, depending on the experimental requirements. This labeling ensures that the instrument can precisely measure fluorescence intensity as the cells pass through the flow cytometer.

Once labeled, the cell mixture flows through the instrument's flow cell in a single file. As each individual cell passes through a laser beam, the detector captures three key parameters: fluorescence, forward-scattered light (FSC), and side-scattered light (SSC). FSC provides information about the cell's size, while SSC gives insights into the cell's internal complexity or granularity. The fluorescent signals provide specific data on the proteins or markers present on the cell surface, allowing further differentiation among cell populations.

FACS instruments are equipped with multiple lasers, filters, and detectors to measure fluorescence across different wavelengths, enabling the simultaneous detection of multiple fluorescent markers. This multi-parameter detection is key for experiments requiring the identification and sorting of complex cell mixtures. The versatility of FACS allows users to tailor their experiments based on specific fluorescent markers and the characteristics of the cells being analyzed.

Once the cell’s properties have been analyzed, the instrument uses electrostatic deflection to physically sort the cells into different containers. Cells that meet specific user-defined criteria are charged and diverted into designated containers. This precise sorting process allows researchers to isolate subpopulations of cells for further analysis or experimental manipulation, making FACS an essential tool for experiments requiring highly specific cell populations.