3.3.2. SYBR Green I Inhibition Assay for the Asexual Stages of P. falciparum

The parasites were synchronized through sterile 5% (m/v) D-sorbitol treatment over 10 min at 37 °C for the enrichment of ring-stage parasites [59]. The cultures were pelleted by centrifugation at 600× g for 5 min. The parasitaemia was determined by microscope analysis of thin blood smears stained with Giemsa 10% after methanol fixation. The initial parasitaemia was calculated from 1000 red blood cells (RBCs), and cultures were diluted to 0.5% parasitaemia and 2% haematocrit by adding the appropriate volumes of erythrocytes and medium. Parasite aliquots of 180 µL were distributed into 96-well plates previously prepared with 20 µL aliquots of a 10-fold concentrated compound. The concentration of the quinoline derivatives ranged from 100 µM to 1.5 µM (two-fold serial dilutions were carried out). Chloroquine was used as positive control and tested in a concentration ranging from 100 nM to 1.5 nM. The tested compounds were diluted to a stock concentration of 20 mM in 100% DMSO before the experiments and maintained at −20 °C. The DMSO concentration in the assay was maintained below 0.5% (v/v). Negative and positive control wells corresponding to non-parasitized erythrocytes and parasite cultures in the absence of compounds were set in parallel. The DMSO concentration was maintained below 0.5% (v/v). The plates were incubated for 72 h at 37 °C in a humidified incubator with a gas mixture of 90% N₂, 5% O₂, and 5% CO₂. After incubation, the culture medium was removed, and the cells were resuspended in 100 µL PBS buffer (116 mM NaCl, 10 mM Na₂HPO₄, 3 mM KH₂PO₄) and lysed with 100 µL lysis buffer (20 mM Tris base, 5 mM EDTA, 0.0008% (v/v) Triton X-100, and 0.008% (m/v) saponin, pH 8.0) containing 0.002% (v/v) SYBR Green I. Plates were incubated at room temperature for 30 min, and the fluorescence corresponding to the parasitic density was determined using a SpectraMAX Gemini EM plate reader (Molecular Devices Corp., Sunnyvale, CA, USA) (excitation at 485 nm, emission at 535 nm). The inhibitory activity of each compound was assessed in duplicate, and the results were compared with the control cultures grown in complete medium with no inhibitors. The half maximal inhibitory concentration (IC₅₀) was determined by non-linear regression analysis of the resulting concentration–response curve using the software Origin 2016 (OriginLab Corporation, Northampton, MA, USA). The reported IC₅₀ values represent the average and standard deviation of two independent experiments.