Uscanga-Palomeque Ashanti C, Osorio Yaneth E, Melby Peter C
Department of Internal Medicine, University of Texas Medical Branch, Galveston, Texas, TX, USA.
Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston; Texas, TX, USA.
Bio Protoc. 2023 Sep 20;13(18):e4817. doi: 10.21769/BioProtoc.4817.
Immune cell trafficking in steady-state conditions and inflammatory cell recruitment into injured tissues is crucial for the surveillance of the immune system and the maintenance of body homeostasis. Tracking the cell journey from the infection site in the skin to lymphoid tissues has been challenging, and is typically determined using fluorescent cell tracers, antibodies, or photoconvertible models. Here, we describe the detailed method to track -infected myeloid cells migrating from the skin to lymphatic tissues by multiparametric flow cytometry. These methods involve labeling of infective Leishmania donovani parasites with fluorescent cell tracers and phenotyping of myeloid cells with fluorescent antibodies, to determine the infection status of migratory myeloid cells. We also describe the detailed protocol to trace donor monocytes transferred intradermally into recipient mice in Leishmania donovani infection. These protocols can be adapted to study skin-lymphoid tissue migration of dendritic cells, inflammatory monocytes, neutrophils, and other phagocytic myeloid cells in response to vaccine antigens and infection. Key features • Cell-tracking of cell-trace-labeled parasites and monocytes from the skin to lymphatic tissues after transference into donor mice. • Identification of migratory cells labeled with fluorescent cell tracers and antibodies by flow cytometry. • Isolation, labeling, and transference of bone marrow monocytes from donor mice into the skin of recipient mice. • Description of a double-staining technique with fluorescent cell tracers to determine cell and parasite dissemination from the skin to lymphoid tissues.
在稳态条件下免疫细胞的迁移以及炎症细胞向损伤组织的募集对于免疫系统的监测和机体稳态的维持至关重要。追踪细胞从皮肤感染部位到淋巴组织的行程具有挑战性，通常使用荧光细胞示踪剂、抗体或光转换模型来确定。在此，我们描述了通过多参数流式细胞术追踪感染的髓样细胞从皮肤迁移至淋巴组织的详细方法。这些方法包括用荧光细胞示踪剂标记感染性杜氏利什曼原虫寄生虫，并用荧光抗体对髓样细胞进行表型分析，以确定迁移性髓样细胞的感染状态。我们还描述了在杜氏利什曼原虫感染中追踪皮内转移至受体小鼠的供体单核细胞的详细方案。这些方案可适用于研究树突状细胞、炎性单核细胞、中性粒细胞和其他吞噬性髓样细胞在响应疫苗抗原和感染时从皮肤到淋巴组织的迁移。关键特性 • 将细胞示踪标记的寄生虫和单核细胞转移至供体小鼠后，从皮肤到淋巴组织进行细胞追踪。 • 通过流式细胞术鉴定用荧光细胞示踪剂和抗体标记的迁移细胞。 • 从供体小鼠分离、标记骨髓单核细胞并将其转移至受体小鼠的皮肤。 • 描述用荧光细胞示踪剂的双重染色技术，以确定细胞和寄生虫从皮肤到淋巴组织的扩散。
