Quantifying FACS Data
FACS data collected by the computer can be displayed in two different ways. What we want to know is how many cells of each color were sorted. In the first example (figure 2), we see the intensity of the green or red fluorescence is plotted on the X-axis and the number of cells with each level of flourescence is plotted on the Y-axis. In this example, there were twice as many red cells sorted as green or unlabeled cells, but the level of light was greater from the green cells than the red cells. This method is best if all cells are either green, red or unlabeled and no cells are labeled both colors.
Figure 2. Quantifying FACS data. This graph shows the number of cells (X-axis) and the level of fluorescence emitted (Y-axis) by the labeled cells. Many different colors can be plotted on this graph, but cells should not be labeled by more than one color.
In figure 3, we see a different way to display the same data. The X-axis plots the intensity of green fluorescence while the Y-axis plots the intensity of red fluorescence. The individual black dots represent individual cells and we are not supposed to count the dots but just look at the relative density of dots in each quadrant. From this graph, we can see there were no cells labeled both red and green (top right) and many cells that were unlabeled (bottom left). The number of green-labeled cells (bottom right) is about the same as the number of unlabeled cells, but the number of red-labeled cells (top left) is about twice that of the other two categories of cells. Again, we can see that the level of fluorescence was higher in the green cells than the red ones. This method of graphing the data is especially useful if cells are present that have been labeled both red and green.
Figure 3. Quantification of FACS data. This graph compares the number of cells labeled by two colors - red (Y-axis) and green (X-axis). The intensity of the emitted light increases as indicated by the arrows. The number of cells at each intensity is shown by the number of dots where each dot represents a single cell. This graph does not work for more than two colors but it works well when individual cells can be labeled by both colors at the same time.
