T. cruzi amastigote proliferation assay

To determine the number of divisions that an intracellular T. cruzi amastigote has undergone in a defined period of time, a modified flow cytometry protocol, based on [34] was performed. Briefly, NHDF were plated at 1.5 x 10⁵ per well in 6 well plates and infected with MOI of 15 for 2 hours using CFSE-stained trypomastigotes. For staining, 5 x 10⁶ trypomastigotes/mL were stained with 1 μM CFSE (Thermo Fisher Scientific) in PBS by incubating at 37°C for 15 minutes. Extra dye was quenched by addition of D-10, and trypomastigotes were pelleted by centrifugation at 2100 g for 10 minutes and incubated in fresh D-10 at 37°C for 30 minutes before infection. At 18 (pre-replication) and 48 (replicative phase) hpi, infected monolayers were trypsinized, washed once in PBS, and cells were lysed to release amastigotes by passing the supernatant 10 times through a 28^1/2G needle. Lysate was fixed by adding paraformaldehyde (Electron Microscopy Sciences) to a final concentration of 1% and incubating 20 minutes on ice. Samples were centrifuged at 300 g for 5 minutes to pellet away host debris, and the supernatant centrifuged at 4000 g for 10 minutes to pellet amastigotes. Pellets were resuspended in PBS with 0.1% Triton X-100 and 0.01 μg/mL DAPI for analysis by flow cytometry. Amastigotes were run on a MACSQuant VYB (Miltenyi Biotec) or LSRII (BD Biosciences) and at least 10,000 events were collected per condition. Data was analyzed using FlowJo 7.6, and amastigotes were discriminated based on size and DAPI staining. Proliferation was modeled using FlowJo 7.6, and CFSE intensity at 18 hpi was set as peak 0 for all samples.