Glass fibre filter

Isotopic drug sensitivity assays were employed as described41 (link) to investigate the susceptibility of P. falciparum to the steroid compounds. The procedure depends on the incorporation of radioactive ³H-hypoxanthine, which is taken up by the parasite as a precursor of purine deoxynucleotides for DNA synthesis43 (link). In 96-well microtitre plates (Nunc^R), a twofold serial dilution of the starting concentration of each pharmacologically active compound to be tested was carried out. Parasites were incubated at a parasitaemia of 0.25% (>70% ring forms) and 1.25% haematocrit in hypoxanthine-free medium. After 48 h, 0.5 μCi ³H-hypoxanthine was added to each well, and the plates were incubated for another 24 h. The cells of each well were then collected on a glass fibre filter (Perkin-Elmer, Rodgau-Jügesheim, Germany), washed, and dried. Their radioactivity in counts per minute was considered to be proportional to the number of parasites in the well. All IC₅₀ values were determined at least in quadruple. IC₅₀ values (drug concentrations that produce 50% reduction in the uptake of ³H-hypoxanthine) were calculated.

For whole-cell binding assays39 (link), M1-CHO cells were collected and 70,000 cells per well were incubated with 0.2 nM [³H]N-methylscopolamine ([³H]NMS; PerkinElmer Inc.) and different concentrations of non-labelled competitor with or without 1 μM FR in assay buffer (HBSS supplemented with 20 mM HEPES; pH 7.0) in a 96-well microtiter plate (Fischer Scientific GmbH) at 28 °C in a final volume of 300 μl for 2 h. For radioligand binding experiments performed with CHO-M1 mebranes39 (link)68 (link), membranes (20–40 μg ml⁻¹) were incubated with 0.2 nM [³H]NMS and different concentrations of non-labelled competitor with or without 1 μM FR in a HEPES buffer (10 mM HEPES, 10 mM MgCl₂, 100 mM NaCl, pH 7.4) at 30 °C in a final volume of 300 μl for 2 h in a 96-well microtiter plate.
Binding experiments were terminated by rapid vacuum filtration through a glass fibre filter (PerkinElmer Inc.) and filter-bound radioactivity was determined by solid scintillation counting. Non-specific binding was determined in presence of 10 μM atropine.

2×10⁵ leukocytes from spleens or LN were stimulated for 72 hr with MPT83 (10 μg/ml) or ConA (3 μg/ml), then pulsed with tritiated (³H)-thymidine (5 μCi/ml; Perkin Elmer, MA) for 6 hr. Cells were transferred to glass fibre filters (Perkin Elmer) using a plate harvester (Wallac, MA), liquid scintillant added (Betaplate scint, Perkin Elmer) and incorporation measured (Perkin Elmer).