Tritiated thymidine 3h tdr

Spleen cells (1.5 × 10⁶/mL) were distributed in a 96-well microplate and cultured in the presence of jArtinM (2.25 nM or 4.50 nM), rArtinM (78.00 nM or 156.00 nM), or ConA (24.5 nM). The cells were cultured for 48 h, and, during the last 12 h of incubation, tritiated thymidine ([3H]-TdR; Amersham Bioscience, Boston, MA, USA) was added to the wells (0.5 μCi/well). Cell proliferation was assessed by measuring [3H]-TdR incorporation, and the results were expressed in counts per minute (cpm).

Mice were sacrificed thirty days after the last immunization and their spleens were extracted under aseptic conditions to be homogenized in RPMI 1640 (Gibco) medium [25 (link)]. Cells were adjusted to a concentration of 4 × 10⁶ viable cells/ml in RPMI medium supplemented (10% heat inactivated fetal bovine serum plus 50 IU/ml penicillin, 50 μg/ml streptomycin, and 0.25 μg/ml amphotericin B) and cultured (100 μl per well) in 96-well microtiter plates (Nunc, Denmark), previously sensitized with 2 or 10 μg/ml recombinant proteins or crude B. abortus RB51 proteins (CBPs), respectively, for 72 h at 37°C and 5% CO₂. CBPs were obtained from 60% methanol inactivated Brucella subjected to a treatment with a hypertonic salt solution (1 M NaCl and 0.1 M sodium citrate) for 24 h; then the bacterial suspension was subjected to sonication for 20 minutes and centrifuged to obtain the supernatant [26 (link)]. After culturing, splenocytes were pulsed with 0.5 μCi tritiated thymidine (³H-TdR) per well (Amersham, Life Science, London, UK) and 8 h later radioactivity incorporated into the DNA was measured using a scintillation counter (Beckman LS 6500, USA). As a lymphoproliferation positive control, 10 μg/ml of concanavalin A (Promega, Madison, WI, USA) was used, while complete RPMI 1640 was used as a negative control. All experiments were done in triplicate.

CD4⁺ and CD8⁺ T cells (1.5 × 10⁶/mL) were distributed in 96-well microplates and incubated for 48 h at 37 °C with ArtinM (1.25 μg/mL), ConA (5 μg/mL), or medium alone. The cells were treated with tritiated thymidine ([₃H]-TdR; Amersham Bioscience, Boston, MA, USA) at 0.5 μCi/well. Cell proliferation was assessed by measuring [₃H]-TdR incorporation and the results were expressed in counts per minute (CPM).