Fluorescence-activated cell sorting (FACS) of live cells separates a population of cells into subpopulations based on fluorescent labeling. Sorting involves more complex mechanisms in the flow cytometer compared to a non-sorting analysis. The fluorescence activated cell sorter allows for analyzing cells based on their protein expression, fluorescence intensity, and other parameters, enabling high cell purity and the study of single cell properties.

FACS plays a crucial role in identifying rare cell populations and is fundamental in cell biology, cancer research, cell therapy, and stem cell research, where precise isolation and analysis of specific cells are essential for advancing scientific understanding and developing new therapies.

Compared to other cell sorting methods, such as magnetic activated cell sorting, FACS offers higher resolution and is particularly effective for analyzing complex cell populations with multiple markers.

Download our flow cytometry application guide

Fluorescence-activated cell sorting (FACS) of live cells

Cells stained using fluorophore-conjugated antibodies can be separated from one another depending on which fluorophore they have been stained with. FACS uses surface markers and cell surface markers to distinguish and sort specific cell populations, including immune cells such as T cells and B cells. For example, a cell expressing one cell marker may be detected using a FITC-conjugated antibody that recognizes the marker, while another cell type expressing a different marker could be detected using a PE-conjugated antibody specific to that marker. In addition to fluorophore-conjugated antibodies, monoclonal antibodies and fluorescent dyes are used to label cells based on specific cell markers and cell surface molecules. This is the fundamental principle of flow cytometry.

Live cell sorting goes one step further:

1. Individual cells are "interrogated" by the laser as in a standard flow cytometer.

2. The machine is set up so that each individual cell then enters a single droplet as it leaves the nozzle tip. This droplet is given an electronic charge depending on the fluorescence of the cell inside it.

3. The cell sorter uses a laser beam to analyze fluorescence intensity and cell size, enabling the separation of single cells and target cells from a heterogeneous mixture. Deflection plates attract or repel the cells accordingly into collection tubes. For example:

   1. A single FITC-stained cell in a single droplet would be given a positive charge and be attracted to the left. Collection tubes to the left would collect all the positively charged FITC-stained cell droplets.
   2. A single PE-stained cell in a single droplet would be given a negative charge and be attracted to the right. Collection tubes to the right would collect all the negatively charged PE-stained cell droplets.

4. Sorted cell populations are then analyzed to ensure successful cell sorting

5. Sorted cells can then be cultured.

Compared to other cell sorting methods, such as magnetic activated cell sorting and microfluidic cell sorting, FACS offers the advantages of simultaneous analysis of multiple parameters and, with spectral flow cytometry, even greater resolution. An automated cell counter can be used to assess cell population and viability before and after sorting.

To maintain cell viability and prevent contamination for subsequent cell culture, consider the following tips:

• Include serum in buffers.

• Avoid using sodium azide in the buffers during staining, as it can be toxic to cells and compromise viability.

• Ensure the experiment is conducted in aseptic sterile conditions to prevent cell contamination.

• Performing intracellular staining before sorting live cells is usually not possible, as permeabilization requires damaging the cell membrane, which would compromise cell viability.

• Handle labeled cells and the cell suspension gently to prevent cell death and maintain healthy cells for further analysis.

Figure 1: A schematic representation of FACS of live cells.