Janus green

After 5 days of exposure to idebenone at doses of 0–0.5 µM, the Alamar blue viability assay (Sigma-Aldrich) was performed. Fluorescence of resorufin was read at wavelengths: 544 nm (excitation) and 590 nm (emission) 3 h after adding reagent to the culture medium (1:10). The results are shown as the ratio (%) of the fluorescence intensity of test samples to the control (untreated) samples measured by Fluoroscan Ascent (FL, Labsystems) plate reader. Data presented on the graphs are normalized to cell number which was obtained by Janus Green (Abcam) staining performed according to the manufacturer’s protocol.

Cells at the three stages of neural differentiation were seeded separately on a 96-well plate covered with Matrigel solution (1:30). Idebenone was added to cells for 5 days. After this time the levels of two mitochondrial proteins was measured with MitoBiogenesis In-Cell ELISA Colorimetric kit (Abcam), according to the manufacturer’s instructions, on cells fixed with 4% PFA. SDHA, mt-COX-1 proteins level was obtained on a Fluostar plate reader OMEGA (BMG Labtech). After washing with PBS, cells were incubated for 30 min in 1X Permeabilization Buffer. Prior to the addition of primary antibodies (anti-SDHA and anti-COX-1), the cells were incubated in 2X Blocking Buffer for 2 h followed by a mix of secondary antibodies conjugated with enzymes: (1) alkaline phosphatase and (2) horseradish peroxidase for 60 min. After this time substrate for alkaline phosphatase was added and absorbance was measured at OD 405 nm wavelength, then the substrate for horseradish peroxidase was added, and absorbance was measured at OD 600 nm wavelength. Changes in SDHA and COX-1 levels were normalized to total cell number measured according to manufacturer’s protocol using Janus Green (Abcam) staining method. SDHA and COX-1proteins levels were shown independently as the absorbance ratio (%) of cell samples treated by idebenone to the untreated control.

After 5 days of exposition of NSC, eNP, and NP to idebenone, ROS level was measured by DCFH-DA (dichloro-dihydro-fluorescein diacetate, Sigma-Aldrich) assay. Cells were incubated with DCFH-DA reagent (1 µM) for 3 h. After this time fluorescent DCF(2′,7′-dichlorofluorescin) was detected by a plate reader Fluoroscan Ascent (FL, Labsystems) at wavelengths: 485 nm (excitation)—538 nm (emission). The results are shown as the ratio (%) of the fluorescence intensity of test samples to the untreated control. Normalization of ROS level results to cell number was obtained using Janus Green (Abcam) staining.