Resazurin

Hepato-carcinoma (HepG2) cells (ATCC®HB-8065TM) were cultured in DMEM low glucose, 10% FCS, 1% Penicillin/Streptomycin (P/S) (all Gibco) at 37 °C and 5% CO₂ in a humidified atmosphere. Cells were split with trypsin (Gibco) when they reached confluency. Monolayers were treated with the FD K1V1V1, FD K1V5V3 and FD K2V3V5 fractions at the indicated concentrations (5, 7.5, 10, 12 and 15%; w/v). Cell proliferation was determined by the resazurin metabolic assay [27 (link)]. After treatment for 2 or 3 days with the indicated concentrations of the three fractions, cells were incubated for 4 h with fresh medium containing 10% of resazurin solution consisting of 0.1 mg/mL resazurin (Sigma-Aldrich) in phosphate buffer saline (PBS) (Gibco). resazurin reduction was measured with a spectrophotometer (Bio-tek instruments) at 570 and 600 nm. A final resazurin value (F.O.D.) was calculated as the difference between the O.D. 570/O.D. 600 nm of the treated sample and that of the negative control (resazurin media incubated for 4 h in the absence of cells). The procedure was carried out for 3 days at the same time, in duplicate.

Reporter cells were seeded (1 × 104 cells/well) in 96-well plate, then treated with indicated concentrations of compounds for 18 h. At the assay time point, resazurin (Cayman Chemical, Ann Arbor, MI, USA) was added to a final concentration of 0.1 mg/mL and further incubated for 4 h at 37 °C. Fluorescence of the reduced resazurin (ex/em: 530/590 nm) was measured from the culture supernatant by using a Synergy HT Multi-Mode Reader (BioTek, Winooski, VT, USA) to determine cell viability. The cells were then harvested for luciferase activity measurements according to the manufacturer’s protocol (Promega Corporation, Madison, WI, USA). Relative luciferase activity was calculated by normalizing luciferase activity to cell viability. For GNMT promoter activity assay treatment, data were presented as the fold to DMSO solvent control; for NRF2-activity assay, DMSO solvent control was used as 100% activity.

Astrocytes-hNS1 were seeded in 96-well plates at a density of 1.5x10⁴ cells/well; the next day, they were infected with ZIKV at 0.1 and 1 MOI. At five dpi, the metabolic activity was analyzed using resazurin (Sigma Aldrich) [40 (link), 46 ], and the production of ROS was determined using 2’,7’-dichlorofluorescein diacetate (DCFH-DA, Cat. No D6883, Sigma Aldrich) [47 (link)]. Briefly, the medium was removed and replaced with DMEM medium without phenol red and supplemented with resazurin (10 μg/ mL final concentration), then the cells were incubated for 2 hours at 37°C. The metabolic activity was determined by monitoring the fluorescence of resorufin (resazurin metabolized compound) in a Synergy H1 microplate reader (Biotek) at 560 nm excitation and 590 nm emission. For the analysis of ROS production, the medium was replaced with DMEM without phenol red and supplemented with 10 μM DCFH-DA; the cells were incubated for 2 hours at 37°C. The fluorescence reading was performed at 480 nm excitation and 530 nm emission. For both assays, 3 to 4 independent experiments were performed with three experimental replicates, and the fluorescence emitted by uninfected cells was normalized as 100% metabolic activity and ROS production, respectively.

Nuclear transcription factor NRF2 activity was evaluated in HacaT normal cells and Huh7 cancer cells according to a previously described method (Mykhailenko et al., 2020 (link)). The cell line HaCaT/ARE (antioxidant response element) was developed using a HaCaT cell line carrying a fragment derived from pGL4.37[luc2P/ARE/Hygro] plasmid and the luciferase reporter gene luc2P. The reporter cells were cultured in DMEM (Gibco BRL, Grand Island, NY, United States) supplemented with penicillin (100 U/ml), streptomycin (100 μg/ml), 10% heat-inactivated FBS (HyClone, Logan, UT, United States), and 100 μg/ml hygromycin at 37°C in 5% CO₂. The cells were seeded (1 × 10⁴ cells/well) in a 96-well plate and treated with the sample for 18 h (single measurement). Resazurin (Cayman Chemical, Ann Arbor, MI, United States, final concentration of 0.1 mg/ml) was added, and the cells were incubated for an additional 4 h at 37°C. To determine cell viability, fluorescence of the reduced Resazurin in the cells' supernatant (ex/em: 530/590 nm) was measured using a Synergy HT Multi-Mode Reader (BioTek, Winooski, VT, United States). The cells were then harvested, and the luciferase activity was measured according to the manufacturer's protocol (Promega Corporation, Madison, WI, United States). The luciferase activity was normalized to cell viability.

MIC assays were conducted as described previously (90 (link)). Briefly, overnight cultures of bacteria were subcultured to mid-log phase (OD₆₀₀, 0.4 to 0.6) before being pelleted and resuspended 1:10 in PBS. Antibiotics were diluted in LB or urine and added to 96-well plates. Bacteria were added at a 1:10 dilution to each well before being incubated under stationary, aerobic conditions at 37°C overnight. To measure metabolic activity, resazurin (Sigma-Aldrich) was added at a concentration of 6.75 μg/mL to each well and plates were incubated for an additional 3 h at 37°C. Fluorescence, indicated by a resazurin-to-resofurin conversion, was measured using an excitation/emission of 550 nm/600 nm on a BioTek Cytation 5 instrument. MIC was determined as the lowest antibiotic concentration at which a >90% reduction in fluorescent signal was observed compared with no-antibiotic controls. MIC breakpoints were obtained from the Clinical and Laboratory Standards Institute (CLSI) (91 ).

ARPE-19 cells were seeded (10,000 cells/well) in flat-bottom 96-well microplates in DMEM/F-12 with GlutaMAX™ supplemented with 1% FBS and antibiotics (100 U/mL penicillin, 100 g/mL streptomycin), at a density of 10,000 cells/well in flat-bottom 96-well microplates. ARPE-19 cells were then incubated with 31.25, 62.5, 125, 500, 1000 or 2000 µM of H₂O₂ (PanReac AppliChem, Ottoweg, Germany) for 24 h. In the experiments to assess the impact of TRAP1 loss in ARPE-19, the cells were transfected twenty-four hours after seeding, as previously described. Cell viability/metabolism was assessed using Resazurin (Invitrogen, Waltham, MA, USA) according to the manufacturer’s instructions. Briefly, the medium was replaced with DMEM/F-12 with GlutaMAX™ containing antibiotics and Resazurin (1 mg/mL) and incubated at 37 °C for 2 h. The fluorescence was measured at Ex/Em = 550/590 nm using Synergy Multi-Mode Reader (BioTek, Winooski, VT, USA). Cells were then washed twice with PBS and fixed with 4% PFA in PBS for 10 min. Thereafter, nuclei were counterstained with DAPI (1/5000; Invitrogen, Waltham, MA, USA) and counted using an automated high-content fluorescence microscope (INCell Analyzer 2000, GE Healthcare, Chicago, IL, USA). The values are presented as a percentage of the control (non-treated cells).

Twenty thousand CaSki cells per well were seeded in a 12-well plate. Drugs were added at 24 h after doxycycline induction. At 72 h after drug treatment, cells were incubated with fresh medium containing 10 μg/ml resazurin (Sigma). resazurin conversion was measured with a Synergy H1 microplate reader (BioTek) with 560-nm excitation and 590-nm emission filters as previously described (80 (link)).