Fluorescence activated cell sorting (FACS) of live cells separates a population of cells into sub-populations based on fluorescent labeling. Sorting involves more complex mechanisms in the flow cytometer than a non-sorting analysis. 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 an FITC-conjugated antibody that recognizes the marker, and another cell type expressing a different marker could be detected using a PE-conjugated antibody specific for that marker. This is the basic task of flow cytometry.
Live cell sorting goes one step further:
- Individual cells are “interrogated” by the laser as in a normal flow cytometer.
- The machine is set up so that each individual cell then enters a single droplet as it leaves the nozzle tip. This drop is given an electronic charge, depending on the fluorescence of the cell inside the drop.
- Deflection plates attract or repel the cells accordingly into collection tubes.
A single FITC-stained cell in a single droplet would be given a positive charge and be attracted to the right.
Collection tubes to the right would collect all the positively charged FITC-stained cell droplets.
A single PE-stained cell in a single droplet would be given a negative charge and be attracted to the left.
Collections tubes to the left would collect all the negatively charged PE stained cell droplets.
- Sorted cell populations are then analysed to ensure successful cell sorting.
- Sorted cells can then be cultured.
Remember: The cells need to remain viable and without contamination for subsequent culture
- Include serum in buffers.
- Avoid sodium azide in the buffers during staining as this can be toxic to cells and compromise the viability.
- The experiment should be undertaken in aseptic sterile conditions to ensure the cells do not become contaminated.
- It is not usually possible to do intracellular staining before sorting of live cells, as the permeabilization requires damage to the cell membrane which would compromise the cell viability.