Infinite m200 pro multimode

The performed analysis included the determination of enzymatic antioxidants (salivary peroxidase (Px, EC 1.11.1.7), glutathione peroxidase (GPx, EC 1.11.1.9), catalase (CAT, EC 1.11.1.6), and superoxide dismutase-1 (SOD, EC 1.15.1.1)), nonenzymatic antioxidants (reduced glutathione (GSH) and uric acid (UA)), and redox status (total antioxidant capacity (TAC), total oxidant status (TOS), and oxidative stress index (OSI)), as well as oxidative damage products (advanced glycation end products (AGE), advanced oxidation protein products (AOPP), and malondialdehyde (MDA)).
Nonenzymatic antioxidants and oxidative damage products were assayed in NWS, SWS, and plasma samples, while antioxidant enzymes were assayed in NWS, SWS, and erythrocytes [11 (link), 15 (link)]. Unless stated otherwise, all reagents were purchased from Sigma-Aldrich (Poland, Germany, or USA). On the day of the biochemical tests, the material was slowly thawed at 4°C. All assays were performed in duplicate samples and standardized to 1 mg of the total protein. The absorbance/fluorescence was measured using a 96-well microplate reader (Infinite M200 PRO Multimode, Tecan).

Transepithelial electrical resistance (TEER) was monitored every other day with an EVOM2 Epithelial voltohmmeter with an EndOhm-6 chamber (World Precision Instruments). TEER values were normalized by the total surface area of the cell monolayer. After 21 days, drug permeability studies were performed using FITC-dextran of 4.4 kDa (FD4) as paracellular marker. Briefly, cells were washed with PBS before adding 200 μL of 0.5 mg/mL FD4 to the apical compartment and 600 μL DMEM without phenol red to the basolateral compartment. Throughout the experiment, cells were incubated at 37 °C. At specific time points, samples were withdrawn from the basolateral compartment followed by media replacement. Retrieved samples were then transferred to a 96-well black plate and their fluorescence was read on a microplate reader (Infinite M200 PRO Multimode, Tecan, Männedorf, Switzerland) at 495/520 nm excitation/emission wavelengths. The apparent permeability coefficient (P_app) was calculated by the following equation,
$$P_{app}=\frac{dQ}{dt}· \frac{1}{A·C_0}$$
where dQ/dt is the flux, A the surface area of the cell culture, and C₀ the initial donor concentration of FD4. The steady state flux was used for the calculation of the P_app value. All experiments were done in at least three independent biological replicates.