Novostar

Cells were seeded into 96 wells plate (Corning; 734–1609) three days before staining. Cells were stained with 0.02μM Myrcludex-B—FITC for 30min at 37°C. Myrcludex B-Fitc intensity was measured using a NOVOstar microplate reader (BMG Labtech GmbH, Offenburg) at λex / λem = 483–14 nm/ 530–30 nm. The option orbital averaging was set at a diameter of 3mm. Background fluorescence was subtracted and results were plotted as percentage of NTCP WT.

Mitochondrial membrane potential was measured by the mitochondrial uptake of 20 μm TMRM in a reaction containing 25 mm succinate. The decline in the buffer TMRM concentration was monitored at 575 nm with fluorescent spectrophotometer (NOVOstar, BMG Labtech). Relative membrane potentials were reported for tspo −/−, tspo +/− mitochondria relative to control +/+ mitochondria assayed in parallel wells.

Mitochondrial and cellular ROS levels were measured by using fluorescent probes in 96 well black plates (Greiner Bio-One, Leipzig, Germany; 655090) as previously described [65 (link),66 (link)]. The cells were cultured and treated as above indicated. To measure mitochondrial ROS levels, at the end of incubation, the medium was changed to Hank’s solution (136.9 mM NaCl, 5.4 mM KCl, 1.3 mM CaCl₂, 3.7 mM NaH₂PO₄, 0.4 mM KH₂PO₄, 4.2 mM NaHCO₃, 0.7 mM MgSO₄, 5.5 mM D-glucose and 10 mM HEPES) containing 5 µM of MitoSOX (stock prepared as 5 mM solution in DMSO) and incubated at 37 °C in the 5% CO₂/air incubator for 10 min. Cellular ROS levels were measured by using the fluorescent probe DCFH-DA in Hank’s solution containing 10 µM of the fluorescent probe (stock prepared as 20 mM solution in DMSO) and incubated at 37 °C in the 5% CO₂/air incubator for 40 min. Then, cells were washed with 0.2 mL of Hank’s solution three times and the fluorescence was measured in a fluorescence plate reader (NOVOstar, BMG LABTECH GmbH, Ortenberg, Germany) with incubation at 37 °C. Filters were Ex = 510 ± 10 nm, Em = 580 ± 10 nm for MitoSOX, and Ex = 510 ± 10 nm, Em = 540 ± 10 nm for DCFH-DA, and readings were performed from the bottom of the plate). As positive control for the technique we used rotenone, a specific inhibitor of mCx-I, for mitochondrial ROS levels, and H₂O₂ for cellular ROS levels.

The intracellular ROS levels were measured with the fluorescent probe DCFH-DA (2′,7′-dichlorofluorescein diacetate) according to previous work [33 (link)]. Two plates were prepared to evaluate the ROS levels in cells that were preincubated with 400 μg/mL AOS for 4 and 24 h or were not preincubated. GES-1 cells (10,000 cells/mL) were seeded in 96-well plates (SPL, TCL group) and incubated for 24 h. A control with only medium and a control with 5 mM of antioxidant N-acetyl cysteine (NAc) were placed onto each plate. After the treatment, the cells were washed with PBS 1x, incubated with 10 μM DCFH-DA for 30 min at 37 °C, washed three times with PBS 1x, and exposed to different H₂O₂ concentrations (50, 100, and 200 μM) for 3 h. Finally, the fluorescence intensity of DCFH-DA was measured by NOVOstar (BMG LabTech, Ortenberg, Germany) with excitation and emission wavelengths of 485 nm and 535 nm, respectively. The fluorescence was expressed as a fold change, considering control with only medium as 1 unit.

CHO (Chinese hamster ovary) cells were obtained from Evrogen company (http://evrogen.ru). The CHO cell lines stably expressing rat TRPV1 was generated using T-Rex System (Invitrogen) according to the manufacturer’s instructions and fluorescent assays were performed using NOVOstar (BMG LABTECH, Germany) as described [31 (link)]. CHO cells stably expressing rat TRPV1 were seeded into black-walled, clear-bottomed 96-well plates at a density of 75,000 cells per well (complete media without antibiotics and containing 1 μg/ml tetracycline to induce channel expression) and were cultured overnight at 37°C. The cells were then loaded with the cytoplasmic calcium indicator Fluo-4AM using Fluo-4 Direct^™ Calcium Assay Kits (Invitrogen) and incubated in the dark at 37°C for 60 minutes and then at 25°C for 60 minutes. The buffer alone (control) or APHC-polypeptides were added to the cells. Changes in cell fluorescence (λex = 485 nM, λem = 520 nM) were monitored before and after the addition of TRPV1 agonist (capsaicin or 2APB). The measurements were performed at pH 7.4 and 25°C.