Wallac microbeta scintillation counter

This assay was performed for competition binding to the colchicine and vinblastine binding sites on tubulin and carried out in a 96-well plate. Biotin-labeled tubulin (Cytoskeleton, Denver, CO) was dissolved in G-PEM buffer (80 mM PIPES pH 6.8, 2 mM MgCl₂, 0.5 mM EGTA, 1 mM GTP and 10% glycerol) to a final concentration of 0.8–1 mg/ml. [³H]colchicine or [³H]vinblastine (final concentration of 50 nM and 100 mM, respectively), 50 µM of SRF and 1.0 µg of Biotin-labeled tubulin was mixed in a final reaction volume of 100 µl of G-PEM buffer and incubated for 45 min at 37°C. Streptavidin SPA beads (80 µg/well) was added to each reaction mix and incubated for an additional 30 min at 4°C. The radioactive counts were measured using a Wallac Microbeta scintillation counter (PerkinElmer, Waltham, MA). For control reactions, SRF was omitted from the reaction mix.

In the first set of the experiments, VP-16 influence on the proliferation activity of normal lymphocytes was tested. For this purpose, lymphocytes, freshly isolated from blood by Ficoll-Paque centrifugation, were re-suspended in RPMI supplemented with 10 % fetal bovine serum (FBS), stimulated with soluble antibodies: anti-CD3 and anti-CD28 (1.0 μg/mL) and then incubated with different VP-16 concentrations. Two experiments with two different times of VP-16 exposition (24 and 72 h) were conducted. In the first experiment, VP-16 was removed after 24 h and subsequently 3-day stimulation of the lymphocytes with the antibodies without VP-16 was performed. In the other experiment, VP-16 was present in the culture medium for all the incubation time (3 days of the antibodies stimulation). The lymphocytes were harvested and beta-radiation after [3H]-thymidine incorporation was counted as a measure of cell proliferation using Wallac Micro Beta scintillation counter (Perkin Elmer).

To test the influence of VP-16 on cytotoxic activity of lymphocytes we used JAM assay. Effector lymphocytes were firstly stimulated and incubated with VP-16 either for 24 or 72 h and mixed with the target tumor cell line in different ratios. In the first experiment, the effector cells were stimulated in vitro with the soluble anti-CD3/CD28 antibodies and cultured with 0.1 μg/mL VP-16 for 3 days. In the other experiment, VP-16 was washed out after 24 h exposition followed by 48 h stimulation with the antibodies. The target cells were P815 mastocytoma cell line which functions in a cell-mediated cytotoxicity manner. One day before the test, the target cells were incubated with [3H]-thymidine (10 mCi per 1 mL) overnight, then coated with the soluble anti-CD3 antibody for 1 h and washed. The assay was performed on V-shaped 96-well plate. The target cells were co-cultured with the stimulated lymphocytes at a particular effector/target ratio (25:1, 12.5:1, 6.25:1, 3:1, 1.5:1, 0.75:1) for 6 h. The lymphocytes were subsequently harvested (using Perkin Elmer Cell Harvester) and the radioactivity was measured using a Wallac Micro Beta scintillation counter (Perkin Elmer).