Microbeta plate counter

T cells were purified from mouse spleen using MACS with a pan T-cell isolation kit (Miltenyi Biotec). A total of 2 × 10⁵ normal splenic T cells were stimulated with 1 μg/ml of anti-CD3 (eBioscience, Waltham, MA) and 10 μg/ml of anti-CD28 (BD Bioscience) antibodies in RPMI 1640 medium containing 10% heat-inactivated FBS and 2 mM glutamine. FACS-sorted CD11b⁺Gr1^hi cells from the LPS + PBS group or the LPS + TDCA group were mixed with T cells at a final concentration of 4 × 10⁴/well (E:T = 1:5) in a 96-well flat-bottom plate (Nunc, Roskilde, Denmark) and cultured for 96 h at 37°C in a humidified 5% CO₂ atmosphere. T cells cultured without CD11b⁺Gr1^hi cells served as a negative control. The cells were pulsed with 1 μCi [³H] methyl-thymidine (Perkin Elmer, Waltham, MA) for 18 h. The cells were harvested with a Filtermate Harvester (Perkin Elmer), and the isotope incorporation was measured using a MicroBeta Plate Counter (Perkin Elmer). The data are expressed as the counts per minute (cpm) ± standard error of the mean (SEM).

To determine whether ΔPbnpt1 parasites were deficient in formation of male gametes (as reported previously14 (link)), microgametogenesis was induced in WT and ΔPbnpt1 parasites by a drop in temperature and the addition of xanthurenic acid. The extent of [³H]hypoxanthine incorporation into DNA that is newly synthesized upon exflagellation of the microgametocytes was measured as described previously54 (link). Briefly, wild type and ΔPbnpt1 parasite cultures were magnet purified using a VarioMACS separation unit and resuspended in hypoxanthine-free RPMI-HEPES (RPMI supplemented with 20 mM HEPES and 4 mM sodium bicarbonate, pH 8.0). Aliquots of purified parasites were transferred into wells of a 96-well plate to a final volume of 200 μl. 25 μl of gametocyte activating media (RPMI-HEPES supplemented with 100 μM xanthurenic acid and 16 μCi ml⁻¹ [³H]hypoxanthine) was added to each well. The plate was incubated for 10 min at room temperature, during which time competent male gametocytes will undergo three rounds of genome replication followed by exflagellation, then frozen at −20 °C. The plate was thawed to lyse the cells, and radiolabelled DNA transferred to filters using a Filtermate Universal Cell Harvester (Perkin Elmer). The samples were read using a MicroBeta plate counter (Perkin Elmer). This provides a quantitative measure for microgametogenesis.

NK Target cells were grown over night in the presence of ³⁵S added to a Methionine-free media (Sigma). Prior to incubation with the effectors, target cells were washed, counted, and 5000 cells/well were plated. For each target, the spontaneous ³⁵S release was calculated from cells which were not incubated with effector cells, and maximum ³⁵S release was calculated by adding 0.1M of NaOH to the target cells. The level of ³⁵S release was measured after 5 hours of incubation with effectors by a MicroBeta Plate Counter (Perkin Elmer).