Infinite m200 multimode reader

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to assess the cytotoxicity of the inhibitors towards host macrophages and was performed following the manufacturer’s instructions. Primary peritoneal macrophages (PECs) extracted from Balb/c mice were incubated with AQP1 inhibitor (Tocris Biosciences, Bristol, UK) (12.5–400 µM) or ABC transporters modulator verapamil (Sigma Aldrich, St. Louis, MO, USA) (6.25–200 µM) in a 96-well plate for 48 h at 37 °C in 5% CO₂. Following this, 25 μL of (5 mg/mL in 1 × PBS) MTT were added to each well and the plate was re-incubated at 37 °C in the dark. Then, 4 h later, all media was removed and 150 μL of Dimethyl Sulfoxide (DMSO) were added to each well, mixed well by pipetting, and incubated for 15 min in the dark. Absorbance was taken at 540 nm on an Infinite M200 multimode reader (Tecan, Switzerland). A decrease in the absorbance at 540 nm indicated a decrease in cell viability.

The day before treatment, MC3T3-E1 cells were seeded into 96-well plates and treated with 0, 5, 10, 20 or 50 μM resveratrol (Sigma-Aldrich, St. Louis, MO, USA) for 24 and 48 h; or with 0, 10, 20 or 50 mg/L P. endodontalis LPS for 24 h, 48 h and 72 h. Mitochondrial activity was assayed with an MTT (3-[4-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) (Sigma-Aldrich) assay. Absorbance at 570 nm was determined in solubilized cells using an Infinite M200 Multimode Reader (Tecan, Männedorf, Switzerland). The cell growth rate was expressed as a percentage of the control.