An In Vitro Assay to Quantify the Intracellular Growth of Parasite in Macrophages

Take two microplates containing coverslips with bone marrow-derived macrophages or BMMs.

Add Leishmania metacyclic promastigotes — the elongated flagellated stage — into one plate and amastigotes — the oval non-flagellated stage — in another plate.

During incubation, the parasites bind to macrophage receptors, triggering receptor-mediated phagocytosis.

The phagosome fuses with the lysosomes and vesicles derived from the endoplasmic reticulum and Golgi apparatus, maturing into a parasitophorous vacuole.

The vacuole's decreased pH triggers promastigote differentiation into amastigote and its replication.

Wash to remove non-internalized parasites.

Fix the infected macrophages and add detergent to permeabilize the cells and parasites.

Introduce a fluorescent nucleic acid dye to stain the macrophage and parasite nuclei.

Mount the coverslips on slides and observe under a fluorescence microscope, quantifying the larger host nuclei and surrounding smaller amastigote nuclei.

Compare amastigote numbers to assess their multiplication within macrophages initially infected with undifferentiated promastigotes versus those infected with replicative amastigotes.