Acapella

Plates were imaged using an Opera QEHS high content screening system with a 20X 0.45NA ELWD Plan Fluor (air) objective (3 image fields/well, ~200 cells/well). The images were analyzed using the PML detection protocol (S1 Fig), which was developed based on a custom Acapella (PerkinElmer) nuclei and spot detection algorithm. A total of 46 analysis parameters were extracted from the images, including 12 parameters related to DAPI-channel nuclei morphology and fluorometry, 12 parameters related to AlexaFluor488 channel cell morphology and fluorometry, 19 parameters related to spot detection under nuclei mask, 1 parameter overall nuclei (cell) count, and 2 parameters derived from nuclei parameters (small & bright nuclei): number of “dying cells”, and percentage “dying cells”. Images and data were uploaded to the Columbus image-data management system (PerkinElmer). For the primary HCS campaign, the “Percentage of PML-Foci Positive Cells” (% of cells with higher than baseline number of foci) was used as primary assay read-out. For confirmation and secondary assays, multiple foci related assay read-outs were evaluated and “well average of number of foci per cell” or “well average of integrated foci staining intensity per cell” were utilized depending on the assay.

To define presence or absence of T. cruzi amastigotes within delimited cell boundaries (cells cytoplasms) the Acapella software (Perkin-Elmer) was used to develop a very selective script based in the algorithms provided by the software´s ‘building blocks’ approach. The script included the following steps (Fig. 1):
$sd(x)=\sqrt{(e{}^{{\sigma }^2}}-1)e^{2\mu +{\sigma }^2}$ , over the expected value $E(x)=e^{\mu +{\sigma }^2/2}$
(The method to determine these thresholds is explained in “Image analysis method” section of Results). The spots eventually counted as true amastigotes are shown in green. It is observed that there are remaining putative spots which contribute to a certain ‘noisy’ background in non-infected cells and BNZ treated ones (see corresponding panels in Fig. 1e).

Quinacrine hydrochloride, pyronaridine tetraphosphate, and tilorone dihydrochloride (BOC Sciences, Shirley, NY), bafilomycin A1, and chloroquine diphosphate (Sigma, St. Louis, MO) were dissolved in either DMSO or water as 10 mM stock solutions and were stored at -20°C. The nucleus staining dye, Hoechst 33342, CellMask Deep™ Red cytoplasmic/nuclear stain, NHS-Alexa-488 dye, the Dual-Glo® Luciferase Assay System and CytoTox 96™ assay kit were purchased from Promega (Promega, Madison, WI). The modified MTT assay Cell Counting Kit 8 was procured from Dojindo Molecular Technologies (Dojindo Molecular Technologies, Gaithersburg, MD). The 96-well high-content imaging plates were obtained from BD (BD Biosciences, Franklin Lakes, NJ) and 96-well white-walled tissue culture plates were from Corning (Corning Life Sciences, MA). The Opera QEHS confocal imaging reader, Acapella™ and Definiens™ image analysis packages were purchased from PerkinElmer (PerkinElmer, USA). Image acquisition was done using Nikon TI eclipse high content imaging enabled microscope running NIS elements high content imaging software (version 4.30.02).