2 2 azinobis 3 ethylbenzothiazoline 6 sulfonic acid (abts)

Within 1 h after the last triggered SD with (n = 12) or without (n = 9) prior TBI, rats were euthanized, and their brains were dissected for antioxidant capacity assays as previously described [76 (link)]. Brain tissue of healthy rats was collected as control (n = 13). The injured right cortical hemisphere was isolated and homogenized (100 mg/mL) in 1.55 M KCl. Brain homogenates were added to a reaction mix with myoglobin (Sigma-Aldrich, St. Louis, MO, USA, ref. M0630) and 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) (Roche, Mannheim, Germany, ref. 10102946001), and ABTS oxidation by H₂O₂ was measured spectrophotometrically (SpectraMax M3, Molecular Devices, San Jose, CA, USA) at an absorbance of 734 nm (Figure S2). Time to absorbance shift was measured and compared to a Trolox (an artificial antioxidant) (EMD Millipore, Temecula, CA, USA, ref. 648471) standard line.

A stock solution of 12 mM of 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS, Sigma-Aldrich, Gyeonggi-do, Korea) and 5 mM of potassium was prepared and then added to a potassium phosphate buffer solution (pH 7.4) at a ratio of 1:1, and it was stored in a dark room for 12 to 16 h. Thereafter, the absorbance was diluted with distilled water to obtain an absorbance of 0.70 at 734 nm before use. The sample (50 µL) and ABTS solution (1000 µL) were added and left for 15 min in a dark room, and the absorbance was measured at 734 nm using a spectrophotometer (SpectraMax iD3, Molecular Devices). The ABTS radical scavenging ability was converted by the following Equation (2).

where A₇₃₄ control is the absorbance of the control at 734 nm, and A₇₃₄ sample is the absorbance of the sample at 734 nm.