Infinity alt reagent

Plasma ALT was measured using the ALT Infinity reagent (Thermo Fisher) following the manufacturer’s instructions.

All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of North Carolina at Chapel Hill. C57BL/6J mice were fasted overnight (12–15 h) to deplete glutathione stores before acetaminophen (APAP) (Sigma) administration. Fresh APAP was dissolved in warm (~50 °C) sterile saline, cooled to 37 °C, and injected intraperitoneally at 400 mg/kg. As a positive control, mice were injected with saline intraperitoneally. Plasma collected 24 h after injection was measured for ALT concentration using the ALT Infinity reagent (Thermo Fisher) following the manufacturer’s instructions.

WT, Ager^AHA/AHA, and Ager^−/− mice were fasted overnight (12–15 hr) to deplete GSH stores before APAP (Sigma-Aldrich) administration. Fresh APAP was dissolved in warm (~50°C) sterile saline, cooled to 37°C, and injected intraperitoneally at 300 mg/kg. The plasma samples were collected 24 hr after APAP administrations for measuring ALT concentration. In general, the concentration of ALT reached its highest point at 24 hr after APAP overdose. For B2 protective treatment, WT mice were injected intraperitoneally with 10 mg/kg of B2-IgG 12 hr before APAP overdose (300 mg/kg). Plasma ALT was measured using the ALT Infinity reagent (Thermo Fisher Scientific) following the manufacturer’s instructions. Livers were harvested, fixed for 24 hr in 10% neutral buffered formalin. Samples were further embedded in paraffin and sectioned at 4 μm for hematoxylin and eosin staining. The necrosis area was quantified with ImageJ.