Imaging-based Assay for T. cruzi

The T. cruzi 48 h image-based assay was performed as previously described [28 (link)]. Briefly, 3T3 mouse fibroblast cells were added at 1 × 10³ cells/well in 50 μL of medium into 384-well Collagen I coated plates (CellCarrier, PerkinElmer, Waltham, MA, USA) using a Multidrop (ThermoFisher Scientific, Waltham, MA, USA). Plates were incubated for 24 h at 37 °C and 5% CO₂ before addition of 10 μL of T. cruzi trypomastigotes at a MOI of 5:1 parasite–host cells. Plates were incubated for 24 h and then wells were washed three times with PBS containing magnesium and calcium, once with additions using a Multidrop (ThermoFisher Scientific, Waltham, MA, USA) and twice using a Bravo liquid handler (Agilent Technologies, Santa Clara, CA, USA), with 50 μL of media replaced after the third wash. The initial wash was carried out by discarding the supernatant into a reservoir containing bleach, under sterile conditions. Five microlitres of prediluted compounds were added to plates with a MiniTrak compound handing device (PerkinElmer, Waltham, MA, USA) and incubated for 48 h before fixing cells with paraformaldehyde and staining with fluorescent nuclear and cytoplasmic markers. Plates were imaged on an Opera image-based system (PerkinElmer, Waltham, MA, USA).