A fluorometric assay to monitor mitogenic stimulation of human lymphocytes

A fluorometric assay using Hoechst 33342 to measure DNA in cell culture has been applied to detect mitogenic stimulation of human lymphocytes. Mononuclear cells from the peripheral blood of healthy donors and of HIV infected patients were stimulated with mitogens (PHA and ConA, respectively) in serum-free culture medium (5 x 10(4) or 1 x 10(5) cells/well) and incubated with Hoechst 33342. Fluorescence intensities were read using an automated fluorescence reader against the dye solution as a blank. The stimulation index (fluorescence units of stimulated vs. unstimulated cell culture) was calculated and compared to the stimulation index obtained in the [3H]thymidine assay. A sensitivity of 94.6% and a specificity of 97.4% was observed for the fluorometric assay. The intra-assay variability (12.3-15.6%) and the interassay variability (9.6-21.8%) were calculated using 1 x 10(5) cells/culture for PHA stimulation. We recommend the inexpensive and rapid fluorometric Hoechst 33342 assay as an alternative procedure, for monitoring mitogen induced stimulation of human lymphocytes in the diagnosis of immunodeficiencies.