Standardization of Tissue Culture Conditions for Spontaneous Thymidine-2-14C Incorporation by Unstimulated Normal Human Peripheral Lymphocytes: Circadian Rhythm of DNA Synthesis

The rate of DNA synthesis by human lymphocytes was studied in vitro by measuring unstimulated thymidine-2-14C incorporation (spontaneous lymphocyte blastogenesis; SLB). Freezing lymphocytes and extracting DNA after thawing did not alter the radioactive label count rate and was as efficient as extracting DNA immediately after culture. Omission of fetal calf serum also did not alter the rate of DNA synthesis. Standards established as optimal for studies of SLB were: cell concentration, 1.0 times 10(6)/ml/tube; 14C-TdR concentration, 0.4 mjCi/tube; duration of incubation, 8 hr. In sets of identical samples obtained by specimen division, the variation in counts was 6%. To achieve reproducibility of results; it was essential to count the lymphocytes, and then to ensure that each tube contained almost precisely known numbers of cells. Diurnal variations in the rate of DNA synthesis by circulating lymphocytes of healthy men were measured in vitro by SLB at 2-hr intervals for 24 hr. Leukocyte counts, hematocrit, hemoglobin, plasma cortisol, and body temperature were monitored concurrently. The DNA synthesis rate varied in a 24-hr cycle with peaks at 10 A.M. and 11:00 P.M.., depressions at 4 A.M. and 4 P.M. The rate was correlated with body temperature and hematocrit level, and inversely related to the absolute eosinophil count.