In multicellular organisms, all the cells are identical in their DNA but the proteins vary tremendously. Therefore, it would be very useful if we could separate cells that are phenotypically different from each other. In addition, it would be great to know how many cells expressed proteins of interest, and how much of this protein they expressed. Fluorescence Activated Cell Sorting (FACS) is a method that can accomplish all these goals.
The process begins by placing the cells into a flask and forcing the cells to enter a small nozzle one at a time (figure 1). The cells travel down the nozzle which is vibrated at an optimal frequency to produce drops at fixed distance from the nozzle. As the cells flow down the stream of liquid, they are scanned by a laser (blue light in figure 1). Some of the laser light is scattered (red cone emanating from the red cell) by the cells and this is used to count the cells. This scattered light can also be used to measure the size of the cells.
If you wanted to separate a subpopulation of cells, you could do so by tagging those of interest with an antibody linked to a fluorescent dye. The antibody is bound to a protein that is uniquely expressed in the cells you want to separate. The laser light excites the dye which emits a color of light that is detected by the photomultiplier tube, or light detector. By collecting the information from the light (scatter and fluorescence) a computer can determine which cells are to be separated and collected.
Figure 1. Diagram of FACS machine. Cells have been fluorescently tagged with either red or green antibodies, though not every cell expresses the epitope and therefore some are not tagged either color.
The final step is sorting the cells which is accomplished by electrical charge. The computer determines how the cells will be sorted before the drop forms at the end of the stream. As the drop forms, an electrical charge is applied to the stream and the newly formed drop will form with a charge. This charged drop is then deflected left or right by charged electrodes and into waiting sample tubes. Drops that contain no cells are sent into the waste tube. The end result is htree tubes with pure subpopulations of cells. The number of cells is each tube is known and the level of fluorescence is also recorded for each cell.
