CHEMBL3431448

GSK_TCAKS: pIC50 Trypanosoma cruzi Imaging: Amastigotes/Cell. H9c2 cells were seeded in T-225 flasks (225cm^2 culture surface; Corning Inc., NY, USA) in DMEM-10% FBS for 4h to allow attachment. Cells were then washed once with PBS before infection. T. cruzi trypomastigotes, collected at days 5 to 8 after infection from LLC-MK2 parasite infected cultures, were allowed to swim out for 4h at 37C from a centrifuged pellet (2,500 rpm/10 min/room temperature). Trypomastigotes were then collected and counted in a CASY Cell Counter. Trypomastigotes, in supplemented DMEM, were added to H9c2 cultures in a multiplicity of infection of 1 and incubated for 18 h. Cells were washed once with PBS before incubation of the infected H9c2 monolayer with trypsin (Life-Technologies) to detach cells from the flask. Cells were counted in a CASY Cell Counter using a 150 um capillary and their density set at 5 * 10^4 cells per mL in supplemented assay DMEM. Infected H9c2 were dispensed into 384-well poly-lysine coated assay plates at 50uL per well using a Multidrop Combi dispenser. After seeding them, the plates were incubated at 37C, 5% CO2 for 72h. Cultures were then fixed and stained by addition of 50 uL of a solution containing 8% formaldehyde and 4 uM DRAQ5 DNA dye (BioStatus, UK) per well. Plates were kept light-protected and imaged one hour later in a Perkin-Elmer Opera microscope using a 20x air objective (NA 0.4) and the following acquisition set: a 635 nm laser excitation line and a 690/50 emission detection filter for DRAQ5 detection. Five images were collected per well for reliable statistical analysis. Automated image analysis was performed with a script developed on Acapella High Content Imaging and Analysis Software (PerkinElmer). Output: number of amastigotes per cell as infection level measurement.