In cell 2000 analyzer bioimaging system

The potential cytotoxicity of 5A was tested by alamarBlue^TM Cell Viability Assay (Thermo Fisher Scientific, Waltham, MA, USA) as previously described [18 (link)]. Briefly, 1 × 10⁶/mL rhesus monkey kidney epithelial cells (LLC-MK2), and 1 × 10⁶/mL BALB/c IPФ were seeded in a 96-well clear bottom black microplate (BD Biosciences, Franklin Lakes, NJ, USA). Cells were incubated in the presence of increasing drug concentrations for 72 h (LLC-MK2) or 48 h (IPФ) at 37 °C, 5% CO₂, followed by addition of alamarBlue^TM. Fluorescence was measured using a fluorometer (Fluoroskan; Thermo Fisher Scientific, Waltham, MA, USA). Compound 5D or 5E were incubated with 1 × 10⁵ cells/mL (LLC-MK and IPФ) for 72 or 48 h, respectively, at 37 °C, 5% CO₂. After the incubation period, a dilution of 20:1000 in PBS from a stock at 1 mg/mL of Propidium Iodide (PI) and Hoechst 33342 (Thermo Scientific, Waltham, MA, USA) were added for survival discrimination as previously described [44 (link)]. Analysis was performed by High-Content Imaging Assay (HCIA) using an IN Cell 2000 Analyzer Bioimaging System (GE Healthcare, Chicago, IL, USA) for LLC-MK2 cells, and BD Pathway 855 High-resolution Bioimager System (BD Biosciences, Franklin Lakes, NJ, USA) for IPФ. The 50% cytotoxic concentration (CC₅₀) and selective index (S.I.) was determined and summarized in Table 1.

BALB/c IPФ were acquired and seeded at a density of 1 × 10⁵ cells/mL for 2 h at 37 °C, 5% CO₂. After adherence, IPФ were infected with 1 × 10⁶/mL metacyclic promastigotes of L. major-luc, at a ratio of 10:1 parasites per macrophage. Subsequently, infected IPФ were incubated with derivatives 5A, 5D and 5E at increasing concentrations (0.625 to 10 µM) for 48 h treatment. Afterwards, cells were fixed with 4% paraformaldehyde, and stained with (1.25:100) Alexa Fluor™ 488 Phalloidin (Thermo Fisher Scientific, Waltham, MA, USA) and (1:1000) DAPI (Sigma Aldrich, St. Louis, MO, USA). Then, the numbers of infected cells and amastigotes were determined by HCIA using an IN Cell 2000 Analyzer Bioimaging System (GE Healthcare, Chicago, IL, USA). Parameters were set for the excitation and emission spectra of Alexa Fluor™ 488 Phalloidin and DAPI, and a constraint of 3 or more parasites per macrophage was set as previously reported [44 (link),45 (link)].