Microbeta trilux liquid scintillation counter

Antimalarial activity of extracts and compounds from D. bulbifera against P. falciparum K1 and 3D7 strains were assessed by measuring ³H-hypoxanthine incorporated in parasite nucleic acids using the modified technique of Desjadins et al. [24 (link)]. The red cell suspension at 1–2% hematocrit containing 1% ring stage P. falciparum-infected red blood cells was incubated with various concentrations of extracts (2.50–80 μg/ml) and pure compounds (2.5–80 μM) in 96-well culture plates. Parasites were incubated at 37 °C for 24 h, then ³H-hypoxanthine (25 μl of 0.025 μCi/μl) was added to each well, and the plates were maintained under conditions of 93% N₂, 3% O₂ and 4% CO₂ at 37 °C for an additional 24 h. The samples were transferred to a glass fiber filter (Wallac, Turku, Finland). ³H-Hypoxanthine uptake was then assessed using a MicroBeta TriLux Liquid Scintillation Counter (PerkinElmer, USA). Each assay condition was performed in triplicate independently. The concentration of samples that inhibited the uptake of ³H-hypoxanthine by 50% (IC₅₀) assessed by log dose response curve using WinNonlin software version 4.1 (Pharsight Corporation, CA) was used as a marker of antimalarial effect potency. Artesunate at concentrations ranging from 0.1–20 nM and chloroquine at concentrations ranging from 2.0–250 nM were used as positive controls.

The standard DNA helicase assay measures the unwinding of α ³²P-labelled DNA fragments from a partial M13-17-mer duplex. The reaction mixture (10 ml) containing 20 mM Tris–HCl (pH 9.0), 8 mM DTT, 2 mM MgCl₂, 2 mM ATP, 10 mM KCl, 4% (w/v) sucrose, 80 mg/ml bovine serum albumin (BSA), ³²P-labelled helicase substrate (1000 counts per minute [cpm]) and 2 ml of parasite protein from each fraction was incubated at 37 °C for 20 min. The reaction was terminated with 10 ml of loading dye (10% ficoll, 50 mM EDTA, 10 mM Tris–HCl, 0.25% xylene cyanol, 0.25% bromophenol blue). The substrate and product were separated by electrophoresis in a 20% non-denaturing polyacrylamide gel containing 0.5 × Tris HCl-boric acid-EDTA (TBE) (5.4 g Tris base, 2.75 g boric acid, and 0.465 g EDTA in 1 L of deionized water) at 92 V for 1 h and 40 min (Mini-Protean II Dual Slab Cell, Bio-Rad). The gel was exposed to X-ray film to identify the location of the radiolabelled product, which was then excised from the gel. The incorporated radioactivity was measured using a MicroBeta TriLux Liquid Scintillation Counter (PerkinElmer, Waltham, Massachusetts).
One unit of DNA helicase activity is defined as the amount of enzyme that unwinds 1% of the DNA helicase substrate in 20 min at 37 °C.