Unifilter 96

pCTBs were cultured in 96-well tissue culture plates. One μCi [³H] thymidine (Amersham; Tokyo, Japan) was added to the pCTB culture medium for 6 h at culture times of 24, 48, 72 and 96 h. HeLa cells (plated at a density of 1 x 10⁴ cells/well) were used as a positive control. Cells were harvested using UniFilter-96 (PerkinElmer Life Sciences; Waltham, MA), and [³H] thymidine uptake was then measured using a liquid scintillation spectrometer (Top-Count NXT; PerkinElmer Life Sciences).

Sheep PBMCs were plated at a density of 3x10⁵ per well in flat bottom 96-well plates in the presence of BEI-inactivated PPRV (equivalent to 1 x 10⁴ pfu per well prior to inactivation) and incubated at 37°C, 5% CO₂ at a 5⁰ angle. In all experiments, cells were cultured with medium or VDS cell lysate as negative control and with Con-A (0.5 µg/ml) as positive control. All cultures were performed in triplicates. ³H -thymidine was added to each well at 5 µCi/ml final concentration on day 5 and incubated overnight. Cells were harvested onto UniFilter 96 filtermat (Perkin-Elmer) using a cell harvester. After drying, scintillation liquid was added and the filtermat were counted using a 1450 MicroBeta Trilux counter (Perkin-Elmer). Data are presented as stimulation index defined as the ratio of incorporated ³H-thymidine in test to control cultures.

Drug sensitivity tests were performed within 24 hours of bleeding, without culture adaptation. Blood samples were washed three times in RPMI 1640 medium (Sigma Aldrich, St. Louis, USA). The modified isotopic microtest technique [14 (link)] was used. The complete RPMI 1640 medium, i.e. supplemented with 10% of human serum type AB (obtained from the blood bank of Souro Sanon University hospital, Bobo-Dioulasso, Burkina Faso), gentamicin (10 μg/ml) and tritiated hypoxanthine (Amersham, little Chalfont, UK), was used for parasites cultivation. Infected erythrocytes were suspended in this medium at a haematocrit of 1.5% and an initial parasitaemia between 0.1% and 0.5%. This suspension was then added in quantities of 200 μl per well to the plates containing the drugs. These were incubated for 48 hours at 37°C in 5% CO_2. After the incubation period, plates were frozen and thawed to lyse the blood cells. Cultures were then harvested (Harvester Unifilter 96, Packard) on fibre glass paper (reader plates Unifilter 96 Perkin Elmer). The strips obtained were dried and transferred to a plastic bag containing 30 μl of scintillation fluid (Perkin Elmer Betaplate Scint, Wallac). The incorporation of tritiated hypoxanthine was measured using a scintillation Beta counter (Perkin Elmer Wallac MicroBeta Trilux, Turku, Finland).