displayed in the form of a two-dimensional scatter diagram or as a contour diagram, where the fluorescence of one dye-labeled antibody is plotted against that of a second, with the result that a population of cells labeled with one antibody can be further subdivided by its labeling with the second antibody (see Fig. A.21). By examining large numbers of cells, flow cytometry can give quantitative data on the percentage of cells bearing different proteins, such as surface immunoglobulin, which characterizes B cells; the T-cell receptor-associated molecules known as CD3; and the CD4 and CD8 co-receptor proteins that distinguish the major T-cell subsets. Likewise, FACS analysis has been instrumental in defining stages in the early development of B and T cells. This technology also played a vital role in the early identification of AIDS as a disease in which T cells bearing CD4 are depleted selectively (see Chapter 13). Advances in FACS technology permit progressively more antibodies labeled with distinct fluorescent dyes to be used at the same time. For experiments aimed at cell analysis, rather than cell sorting, machines with four lasers that can simultaneously measure 18 different fluorescent dyes are currently available. However, FACS analysis is constrained by the spectral properties of the fluorescent dyes used for coupling to antibodies, and this technology may have reached its limit.
Dot plots IgD IgD IgM IgM Contour maps standard 5% probability color density 0.1 1 10 100 1000 0.1 1 10 100 1000 0.1 1 10 100 1000 0.1 1 10 100 1000 0.1 1 10 100 10000.1 1 10 100 1000 1000 100 10 1 0.1 1000 100 10 1 0.1 Analysis of cells stained with labeled antibodies forward scatter side scatter red PMT green photomultiplier tube (PMT) Laser stream of fuid containing antibody-labeled cells CPU Mixture of cells is labeled with fuorescent antibody with outliers
The magnetic ÿeld is removed, releasing the coupled cells When a magnetic ÿeld is applied, the coupled cells stick to the iron wool; unlabeled cells are washed out N N Heterogeneous population of lymphocytes is mixed with antibodies coupled to paramagnetic particles or beads and poured over an iron wool mesh
An alternative to FACS is a technology based on detecting heavy-metal atoms that are coupled to antibodies. Cell populations labeled with heavy metal-coupled antibodies are analyzed on a machine called a CyTOF™, which combines liquid fluidics with a mass spectrometer. As each cell is analyzed, the quantity of each heavy metal associated with that cell, and thus the abundance of the target of each antibody, is measured. In total, this technology is estimated to have the capability to measure 100 distinct heavy metals, greatly expanding the range of analysis that is currently possible by FACS. However, with this technique, the cells are destroyed by the ionization process required for the mass spectrometry analysis, so the CyTOF cannot function as a cell sorter.
A-19 Lymphocyte isolation using antibody-coated magnetic beads.