Cells are first incubated with a fluorescent dye such as carboxyfluorescein succinimidyl ester (CFSE). This dye enters the cell and, once in the cytosol, becomes covalently coupled to lysine residues on cellular proteins. Each time the cell divides, the amount of CFSE is diluted by one-half, as each daughter cell inherits one-half of the CFSE-labeled proteins. Cell division can then be analyzed by flow cytometry, where a histogram of CFSE fluorescence displays a series of peaks, each of which represents cells that have undergone a fixed number of divisions. Under optimal conditions, this assay is capable of detecting up to 7–8 cell divisions, after which CFSE fluorescence can no longer be measured.
dissociation rate calculated. A new solution of the ligand at a different concentration can then be streamed over the chip and the binding once again measured. The affinity of binding can be calculated in a number of ways in this type of assay. Most simply, the ratio of the rates of association and dissociation will give an estimate of the affinity, but more accurate estimates can be obtained from the measurements of the binding at different concentrations of ligand. From measurements of binding at equilibrium, a Scatchard plot will give a measurement of the affinity of the receptor–ligand interaction.
A-26 Assays of lymphocyte proliferation.
To function in adaptive immunity, rare antigen-specific lymphocytes must proliferate extensively before they differentiate into functional effector cells in order to generate sufficient numbers of effector cells of a particular specificity. Thus, the analysis of induced lymphocyte proliferation is a central issue in their study. It is, however, difficult to detect the proliferation of normal lymphocytes in response to specific antigen because only a minute proportion of cells will be stimulated to divide. Great impetus was given to the field of lymphocyte culture by the finding that certain substances induce many or all lymphocytes of a given type to proliferate. These substances are referred to collectively as polyclonal mitogens because they induce mitosis in lymphocytes of many different specificities or clonal origins. T and B lymphocytes are stimulated by different polyclonal mitogens (Fig. A.32). Polyclonal mitogens seem to trigger essentially the same growth response mechanisms as antigen. Lymphocytes normally exist as resting cells in the G0 phase of the cell cycle. When stimulated with polyclonal mitogens, they rapidly enter the G1 phase and progress through the cell cycle. In most studies, lymphocyte proliferation is most simply measured by the incorporation of 3H-thymidine into DNA. This assay is used clinically for assessing the ability of lymphocytes from patients with suspected immunodeficiencies to proliferate in response to a nonspecific stimulus.
An alternative to the use of a radioisotope to measure lymphocyte proliferation is to use a fluorescent assay that can be performed by FACS. For this assay, the lymphocytes are incubated with a fluorescent dye such as carboxyfluorescein succinimidyl ester (CFSE). This dye enters the cell and, once in the cytosol, becomes covalently coupled to lysine residues on cellular proteins. Each time the cell divides, the amount of CFSE is cut in half, as each daughter cell inherits one-half of the CFSE-labeled proteins. When a population of dividing cells is analyzed by FACS, peaks of CFSE fluorescence can be detected, each of which represents cells that have undergone a fixed number of divisions (Fig. A.33). This assay is capable of detecting up to 7–8 cell divisions, after which CFSE fluorescence can no longer be measured.
Culture cells with antigens A or B antigen A no antigen antigen B Measure T-cell proliferation antigen A antigen B no antigen 53 Proliferation Days of culture After 5–10 days, remove cells from lymph node Immunize with antigen A