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.

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. 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. 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. 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.

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.

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