The incorporation of [3H]-methyl thymidine (3H-TdR) into newly synthesized DNA by lymphocytes incubated with mitogenic lectins may not be an accurate reflection of the number of lymphocytes actually stimulated. To circumvent the problems associated with 3H-Tdr incorporation in lectin-activated lymphocytes, the incorporation of [3H]-thymidine triphosphate ([3H]-TTP) into DNA in human peripheral blood lymphocytes made permeable by cold, hypotonic shock was examined. Ficoll/Hypaque-purified human peripheral blood lymphocytes, incubated in the presence or absence of optimal mitogenic concentrations of phytohemagglutinin (PHA) or concanavalin A (con A), were made permeable by incubation for 15 min at 4°C in a buffer of 10 mM Tris-HCl (pH 7.8), 1 mM EDTA, 30 mM 2-mercaptoethanol, and 4 mM MgCl2. These cells incorporated [3H]-TTP into DNA when incubated in the presence of ATP, dATP, dCTP, and dGTP. The incorporation was linear with cell number and with time for 30 to 60 min in both stimulated and unstimulated lymphocytes. The incorporation was blocked by deletion of any one of the nucleotide triphosphates, or by agents which are known inhibitors of DNA synthesis. The newly synthesized DNA ranged from 4 × 106 to 2 × 108 daltons in size, as measured by centrifugation on alkaline sucrose gradients and was the product of true, semiconservative replication, as determined by CsCl density gradient centrifugation of DNA synthesized in the presence of bromodeoxyuridine triphosphate. The assay has been applied to the measurement of DNA synthesis by a human T cell tumor line and to lectinstimulated murine lymphocytes as well as to human lymphocytes.
Because this method 1) circumvents potential variations in nucleotide transport, metabolism, and pool size, 2) allows direct measurement of DNA synthetic capacity, and 3) provides a means of comparing the DNA synthetic responses of different cell populations or cells from different species to a given stimulus, we suggest that this method is generally applicable to the assay of stimulated lymphocytes and may provide a more accurate measure of lymphocyte activation.