Tbars assay kit

The SK-N-MC cells were plated at a density of 2 × 10⁵ cells/well in six-well plates and treated as described above. The protein concentration was determined with the BCA protein assay kit with BSA (Thermo Scientific, Rockford, IL, USA). SOD activity and MDA level (SOD assay kit and TBARS assay kit, Cell Biolabs, San Diego, CA, USA) were measured at wavelengths of 450 and 532 nm, respectively, in a microplate reader. The GSH level (GSH/GSSG ratio detection assay kit, Abcam, Cambridge, UK) was detected at the Ex/Em of 490 nm/520 nm.

Lipid peroxidation was measured in mice liver tissues using a Thiobarbituric Acid-Reactive Substances (TBARS) assay kit according to manufacturer instructions (Cell Biolabs, France). To prevent lipid oxidation during the assay 100 mg liver tissue was homogenized in PBS (10%) containing 1X BHT. The homogenate was centrifuged at 10 g and 4 °C for 5 min. Then, 100 μL of the supernatant fraction was vigorously mixed with 100 μL of SDS lysis solution and incubated at room temperature for 5 min. After incubation, the thiobarbituric acid (TBA; 250 μL) was added to the mixture and incubation was carried out again, this time at 95 °C for 60 min. After the incubation time, samples were cooled for 5 min in an ice bath and then centrifuged at 3000 rpm for 15 min. Supernatants were collected. To prevent hemoglobin interference in samples, butanol (300 μL) was added to each unknown MDA supernatant (300 μL). The latter solution was vigorously mixed and centrifuged at 10 g for 5 min. Final butanol fractions were transferred to a microplate and absorbance was measured at 532 nm using a microtiter plate reader. TBARS concentrations were expressed as n moles of malondialdehyde (MDA) per milligram of tissue using an MDA standard.

The amount of MDA in the mouse liver tissue was analyzed spectrophotometrically using a thiobarbituric acid-reactive substances (TBARS) assay kit (Cell Biolabs, Inc., San Diego, CA, USA), as suggested by the manufacturer’s instructions.

The RF/6A cells were seeded on six-well plates and treated as above. Catalase (CAT) and Glutathione (GSSH/GSH) activities were observed in oxLDL-loaded cells treated with Etifoxine or XBD173 in media containing 0.1% DMSO and with their control (media containing 0.1% DMSO) by using OxiSelect Catalase Activity Assay Kit (Cell Biolabs, STA-341, San Diego, CA, USA) and the OxiSelect total Glutathione Assay Kit (Cell Biolabs, STA-312, San Diego, CA, USA), following the manufacturer’s guidelines. Similarly, malondialdehyde was quantified using the TBARS assay kit (Cell Biolabs, STA-330, San Diego, CA, USA) according to the manufacturer’s protocol.

The plasma levels of ROS and MDA were determined using the OxiSelect in vitro ROS/RNS Assay Kit (ROS quantification) and TBARS Assay Kit (MDA quantification) according to the manufacturer's instructions (Cell Biolabs Inc, San Diego, USA).