Colorimetric fluorometric assay kit

The fresh root tissue pre-ground in liquid nitrogen to a fine powder was homogenised in 4.5% perchloric acid (PCA). The mixture was briefly vortexed and incubated on ice for 5 min, followed by centrifugation at 15,000× g for 2 min. The excess PCA in supernatant was precipitated with 2 M ice-cold KOH, with the pH being adjusted to 7.75, and the supernatant was re-centrifuged at 15,000× g for 10 min. ATP was determined fluorometrically by glycerol phosphorylation (λ_ex = 535 nm, λ_em = 587 nm) using the Colorimetric/Fluorometric Assay Kit (Sigma-Aldrich).

Plasma creatinine and blood urea nitrogen (BUN) levels were analyzed using a creatinine assay kit (BioAssay Systems, Hayward, CA, USA) and a BUN assay kit (Thermo Fisher Scientific, Waltham, MA, USA), respectively, according to the manufacturer’s protocol. A creatinine value more than 0.5 mg/dL [19 (link)] or a BUN value more than 33 mg/dL [20 (link)] was considered as acute renal failure. Plasma TNF-α and interleukin-6 (IL-6) levels were measured using standard quantitative sandwich ELISA kits (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s protocol. Renal malondialdehyde (MDA) levels were measured using a colorimetric/fluorometric assay kit (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s protocol. Renal levels of reduced glutathione (GSH) and oxidized glutathione (GSSG) were analyzed using a colorimetric detection kit (Enzo Life Sciences, Farmingdale, NY, USA) according to the manufacturer′s protocol.

Renal MDA levels were measured using a colorimetric/fluorometric assay kit (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. The GSH/GSSG ratio was assessed using the Glutathione Detection Kit (Enzo Life Sciences, Farmingdale, NY, USA) according to the manufacturer′s protocol.