Double Staining with Fluorescent Tracers to Determine Myeloid Cell Migration of Leishmania-infected Cells from Mouse Skin to Lymphatic Tissues by Flow Cytometry

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 Leishmania-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.