The next step in the analysis of lymphocyte proliferation (152.7)

Abstract

Abstract The mixed lymphocyte reaction (MLR), in which lymphocytes from two donors are combined and co-cultured, has significantly contributed to our understanding of immune function. Initially, the proliferation of responder cells was evaluated by measuring the uptake of radioactive 3H-thymidine. As technology improved, this method gave way to a technique measuring the incorporation of 5'-bromo-2'-deoxyuridine. Further advances led to assays for detection of incorporated 5'-ethynyl-2'-deoxyuridine and dye-dilution assays using carboxyfluorescein-diacetate succinimidyl ester (CFSE). Here we evaluate another advance in the measurement of lymphocyte proliferation kinetics using CFSE in combination with immunophenotyping and a novel fluorescent cell proliferation tracing dye. In the current study mononuclear cells were isolated from the peripheral blood of healthy human donors using a density gradient and labeled with CFSE or a novel violet cell proliferation dye. One-way MLRs were performed by inhibiting proliferation of antigen-presenting cells with Mitomycin-C. Two-way MLRs were also performed by co-culturing lymphocytes from allogeneic donors. The phenotype, viability, and proliferative index of stimulator and responder cells were evaluated using multicolor flow cytometry.