T sod assay kit

Mito-TEMPO and MitoSOX were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against CHOP, BiP, GRP94, β-actin, and HRP-conjugated secondary antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against SOD2 and caspase-12 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against Sirt3 and acetyl-K68-SOD2 were obtained from Abcam (Cambridge, MA, USA). T-SOD assay kit, SOD2 assay kit, MDA assay kit, ATP assay kit, and mitochondrial isolation kit were purchased from Beyotime Institute of Biotechnology (Jiangsu, China).

The mMECs were cultured overnight in 6-well plates and treated with NAC 1 h prior to S. uberis challenge (MOI 5:1). Total superoxide dismutase (T-SOD) and malondialdehyde (MDA) activity were measured at 6, 12 and 24 h post challenge. The T-SOD activity was measured using a T-SOD assay kit in a microplate reader (Beyotime Biotechnology), whereas MDA activity was evaluated with an MDA ELISA kit (Abcam, Shanghai, China). The same kits were used to determine the activities of T-SOD and MDA in murine mammary glands. Herein, tissues were collected, washed with PBS and stored at −20 °C. Then, tissues were transferred into a mortar with liquid nitrogen, ground to a powder, homogenized with 0.5% Triton X-100 and centrifuged at 14,000× g for 5 min at 4 °C to collect supernatants.