Datta Arani, Barrie Umaru, Wetzel Dawn M
Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX, USA.
Medical Scientist Training Program, University of Texas Southwestern Medical Center, Dallas, TX, USA.
Bio Protoc. 2024 Jun 5;14(11):e5009. doi: 10.21769/BioProtoc.5009.
Leishmaniasis, a neglected tropical disease, is caused by the intracellular protozoan parasite . Upon its transmission through a sandfly bite, binds and enters host phagocytic cells, ultimately resulting in a cutaneous or visceral form of the disease. The limited therapeutics available for leishmaniasis, in combination with this parasite's techniques to evade the host immune system, call for exploring various methods to target this infection. To this end, our laboratory has been characterizing how is internalized by phagocytic cells through the activation of multiple host cell signaling pathways. This protocol, which we use routinely for our experiments, delineates how to infect mammalian macrophages with either promastigote or amastigote forms of the parasite. Subsequently, the number of intracellular parasites, external parasites, and macrophages can be quantified using immunofluorescence microscopy and semi-automated analysis protocols. Studying the pathways that underlie uptake by phagocytes will not only improve our understanding of these host-pathogen interactions but may also provide a foundation for discovering additional treatments for leishmaniasis. Key features • This protocol visualizes and quantifies multiple intracellular forms of . • It offers flexibility at various points for researchers to introduce modifications according to their study needs.
利什曼病是一种被忽视的热带疾病，由细胞内原生动物寄生虫引起。通过白蛉叮咬传播时，该寄生虫结合并进入宿主吞噬细胞，最终导致皮肤型或内脏型疾病。利什曼病可用的治疗方法有限，再加上这种寄生虫逃避宿主免疫系统的技巧，需要探索各种针对这种感染的方法。为此，我们实验室一直在研究吞噬细胞如何通过激活多种宿主细胞信号通路来内化该寄生虫。我们在实验中经常使用的这个方案，描述了如何用该寄生虫的前鞭毛体或无鞭毛体形式感染哺乳动物巨噬细胞。随后，可以使用免疫荧光显微镜和半自动分析方案对细胞内寄生虫、细胞外寄生虫和巨噬细胞的数量进行定量。研究吞噬细胞摄取该寄生虫的潜在途径不仅将增进我们对这些宿主 - 病原体相互作用的理解，还可能为发现利什曼病的其他治疗方法提供基础。关键特性 • 本方案可观察并定量该寄生虫的多种细胞内形式。 • 它在多个环节为研究人员提供了灵活性，以便他们根据研究需要进行修改。
