ANTIBODY-BASED TECHNIQUES - A Guide to Methods in the Biomedical Sciences - page 82

Figure 8. Fluorescence activated cell sorting plots
immunofluorescence. Both methods are highly sensitive and can de-
tect as few as a few hundred to a thousand molecules on an individual
cell.
Figure 8 shows one example of a FACS analysis. Spleen cells were
run through a FACS analyzer and the lymphocyte gate was identified by
forward and side scatter characteristics. The remainder of the analysis
was performed on this isolated gate (by gating, you have the ability to
exclude all other cells in a population from your analysis). The lympho-
cytes were stained with three different antibodies, each with a different
fluorescent probe. The first detects B lymphocytes (B220) in the spleen
cell populations (the other major type of lymphocyte is the T cell). The
B220-positive B cells were then analyzed for the presence of two other
cell surface molecules, CD21 and CD23, that can further divide B cells
into functionally distinct populations (3 different populations are identi-
fied). Note two different types of FACS profiles are illustrated here. The
left panel shows a histogram which discriminates between cells that
highly express B220 and those that do not, while the right panel shows
a contour plot in which cells are monitored by two different antibodies.
The level of expression of a particular molecule is displayed on a log
scale. Current FACS instruments can discriminate label intensities over
a 4 log scale.
Using this type of an analysis, investigators can iteratively begin to
identify smaller and smaller subsets of cells. With sophisticated instru-
mentation and accomplished users, investigators with the right collection
of antibodies can isolate cells that are present at frequencies as low as
1 in 1,000 to 1 in 10,000 cells with high fidelity.
While the description so far deals with the analytical aspects of a FACS
instrument, FACS analysis is coupled with cell sorting technology that
allows an investigator to isolate individual populations of cells. Therefore,