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

To infect the cells with L. Amazonensis, dilute the parasite suspension to the appropriate experimental multiplicity of infection, and add 50 to 100 microliters of parasites to each well of all 6-well plates, including all time points of the experiment. Incubate the bone marrow-derived macrophages for the appropriate infection period and temperature. Then, wash the wells three times with 2 milliliters of fresh 37 degrees Celsius PBS without calcium or magnesium per well, and fix the initial time point samples with 1.5 to 2 milliliters of 2% paraformaldehyde for 10 minutes.

For the plates corresponding to the later time points, add 2 milliliters of fresh bone marrow-derived macrophage medium to each well and return the plates to the appropriate incubator. Then, fix and wash the cells for each remaining time point as just demonstrated, and store the samples at 4 degrees Celsius in fresh PBS.

To stain the cells with DAPI, replace the supernatant with 1.5 milliliters of fresh PBS without calcium and magnesium, supplemented with 0.1% non-ionic detergent for 10 minutes at room temperature, followed by three 2-milliliter washes with PBS without calcium or magnesium. Next, stain the cells with 2 micrograms per milliliter of DAPI in 1 milliliter of PBS for 1 hour at room temperature.

After three washes, place the coverslips cell-side down onto individual glass microscope slides mounted with a commercially available anti-fade mounting reagent. Then, to quantify the number of infected cells, use the 100x immersion oil objective of a fluorescence microscope and Emanuel counter, to identify the number of smaller amastigote nuclei clustered around each large DAPI-stained macrophage nucleus.