GUIDELINE
- 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 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 a FITC-conjugated antibody that recognizes the marker, and another cell type expressing a different marker could be detected using a PE-conjugated antibody specific to that marker. This is the basic task of flow cytometry.
METHODS
- Individual cells are "interrogated" by the laser as in a normal flow cytometer.
- The machine is set up so that each cell 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.
- Sorted cell populations are then analyzed to ensure successful cell sorting.
- Sorted cells can then be cultured.
NOTES
- 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 live cells, as the permeabilization requires damage to the cell membrane which would compromise the cell viability.
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For research use only. Not for any other purpose.
