Begin with a multi-well plate containing bone marrow-derived macrophages on coverslips.
Treat the macrophages with Leishmania promastigotes — the parasite's infectious stage.
During incubation, the parasites interact with macrophages, facilitating the assembly of F-actin around the interaction site.
Macrophages engulf the parasites in F-actin-surrounded phagosomes, forming parasitophorous vacuoles or PVs — a replicative niche.
Within PVs, parasites release survival factors, increasing F-actin accumulation around the vacuoles and inhibiting their fusion with lysosomes.
Additionally, parasites upregulate the production of hepcidin — a macrophage peptide hormone, inhibiting iron release and elevating intracellular iron levels.
The iron influx into vacuoles promotes promastigote differentiation into pathogenic amastigotes and facilitates their multiplication, enhancing virulence.
Post-incubation, wash to remove free parasites, then, fix and permeabilize the infected macrophages.
Introduce a nucleic acid fluorescent dye to stain the nuclei of infected macrophages and amastigotes.
Under a fluorescence microscope, small nuclei clustered around the macrophage's large nucleus confirm the presence of intracellular parasites, demonstrating Leishmania's virulence.
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