Dibello Estefania, Comini Marcelo A, Benítez Diego
Group Redox Biology of Trypanosomes, Institut Pasteur de Montevideo, Montevideo, Uruguay.
Laboratorio de Síntesis Orgánica, Departamento de Química Orgánica, Facultad de Química, Universidad de la República, Montevideo, Uruguay.
Methods Mol Biol. 2022;2524:149-162. doi: 10.1007/978-1-0716-2453-1_11.
This chapter introduces a simple and robust in vitro viability assay to screen bioactive small molecules (e.g., natural, synthetic) against the monomorphic and infective (bloodstream) form of Trypanosoma brucei brucei. The assay relies on a bioluminescent transgenic parasite harboring a genetically encoded copy of a thermostable redshifted firefly luciferase from Photinus pyralis.The major advantages of the assay are simplicity and cost efficiency, along with excellent quality parameters. The bioassay allows estimating parasite numbers and viability (and metabolic state) as a function of bioluminescence (BL) signal. Parasites are grown in the presence of the molecules of interest in a 96-well microplate, and 24 h later, BL is determined with a simple protocol lacking washing steps, using cost-efficient reagents with a reasonable readout time for high-throughput applications.
本章介绍了一种简单且可靠的体外活力测定方法，用于筛选针对布氏布氏锥虫单形感染性（血流）形式的生物活性小分子（如天然、合成小分子）。该测定方法依赖于一种生物发光转基因寄生虫，其携带来自萤火虫萤光素酶的遗传编码的热稳定红移拷贝。该测定方法的主要优点是简单、成本效益高，以及具有出色的质量参数。生物测定法允许根据生物发光（BL）信号估计寄生虫数量和活力（以及代谢状态）。寄生虫在含有感兴趣分子的96孔微孔板中生长，24小时后，使用简单的方案（无需洗涤步骤），采用成本效益高的试剂，并在合理的读出时间内进行高通量应用，测定生物发光。
