Biotek synergy h1

Total antioxidant capacity was measured as the combined antioxidant activities of all serum constituents, including vitamins, proteins, lipids, glutathione, and uric acid. The assay relied on the ability of antioxidants in the sample to inhibit the oxidation of ABTS (2,2′-Azino-di-[3-ethylbenzthiazoline sulphonate]) to ABTS•+ by metmyoglobin and was quantified as Trolox, a water-soluble tocopherol analogue, equivalents. The decrease in ABTS oxidation was quantified colorimetrically at 734 nm using a Biotek Synergy H1 spectrophotometer (Agilent, Santa Clara, CA, USA).
Reduced glutathione (GSH), oxidized glutathione (GSSG), and total glutathione in serum samples were quantified in duplicate using fluorometric GSH/GSSG ratio detection assay kit (Abcam, Cambridge, MA, USA) at the excitation/emission of 490/520 nm (Biotek Synergy H1).

To measure the intracellular NADH/NAD⁺ ratio, NAD/NADH‐GloTM Assay kit (Promega, WI, US) was utilized. Briefly, the cell broth was prepared by cultivating to exponential phase using production medium. The culture broth was mixed with DTAB solution for 5 min. For measuring NADH, 0.4 N of HCl was added to the reaction mix, whereas for measuring NAD⁺, nothing was added. The sample was heated to 60°C for 15 min and cooled to 25°C. The HCl/Trizma solution was added to the reaction mix for NADH, whereas Trizma solution was added to that for NAD⁺. The prepared solutions were mixed with detection reagent at a ratio of 1:1 (v/v) and incubated for approximately 30 min. The luminescence was measured using microplate reader Biotek Synergy H1 (Biotek, VT, USA). The same methods were applied for measuring NAD(H) standard solution. The NADH and NAD⁺ were quantified, and the ratio of them was calculated.
For measuring the intracellular ATP content, BacTiter‐GloTM kit was utilized (Promega, WI, USA). Briefly, the cell broth was prepared by flask culture. The cell broth in exponential phase was taken and washed using distilled water. The same volume of cell broth and reaction mix was resuspended together and rested for 5 min in ambient condition. The luminescence was measured using microplate reader Biotek Synergy H1 (Biotek). The same methods were applied for measuring the ATP standard solution.

Circulating levels of tH3, H3K27ac, and H3K4me3 were measured using enzyme-linked immunosorbent assay (ELISA) kits (Epigentek; Farmingdale, NY) and read on BioTek Synergy H1 (BioTek Instrument Inc., Winooski, VT) at 450 nm with 570 nm correction. CRP was also measured with an ELISA kit (Sino Biological; Wayne, PA) and read on BioTek Synergy H1 (BioTek Instrument Inc., Winooski, VT) as specified above. TNFα, IL-6, and HSP-60 were measured using a multiplex assay (Thermo Fisher, Philadelphia, PA) and analyzed on 3D FlaxAmp (Thermo Fisher, Philadelphia, PA).