Zilberstein Dan, Nitzan Koren Roni
Faculty of Biology, Technion-Israel Institute of Technology, Haifa, Israel.
Methods Mol Biol. 2019;1971:1-8. doi: 10.1007/978-1-4939-9210-2_1.
This chapter describes, in detail, the method our laboratory developed to differentiate L. donovani promastigotes into amastigotes in a host-free culture. This method is based on previous observations that Leishmania promastigotes can combine two environmental signals, typical to lysosomes, acidic pH (~5.5) and body temperature (37 °C), into a signal that induces differentiation. Based on this concept, we have modified medium 199 to make it into an amastigote-specific medium. Shifting promastigotes to this medium, followed by incubation in a CO incubator, induced differentiation. Axenic amastigotes reach maturation within 5 days, resembling the time it takes in vivo. This chapter provides a complete protocol that should be useful for both Old and New World species of Leishmania.
本章详细描述了我们实验室开发的在无宿主培养中将杜氏利什曼原虫前鞭毛体分化为无鞭毛体的方法。该方法基于先前的观察结果，即利什曼原虫前鞭毛体可以将溶酶体特有的两种环境信号——酸性pH（约5.5）和体温（37°C）——组合成一种诱导分化的信号。基于这一概念，我们对199培养基进行了改良，使其成为一种无鞭毛体特异性培养基。将前鞭毛体转移到这种培养基中，然后在二氧化碳培养箱中孵育，可诱导分化。无菌无鞭毛体在5天内成熟，类似于在体内所需的时间。本章提供了一个完整的方案，对旧世界和新世界的利什曼原虫物种都应该有用。
