Fluorescence-activated cell sorting of live cells
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A description of fluorescence-activated cell sorting of live cell populations.
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.
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:
- 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 droplet is given an electronic charge depending on the fluorescence of the cell inside it.
- Deflection plates attract or repel the cells accordingly into collection tubes.
For example:
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.
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. - Sorted cell populations are then analyzed to ensure successful cell sorting.
- 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 this 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.