T bhp

Articular chondrocytes were seeded at 1.5 × 10⁴ cells/well on 96-well plates and transfected with siHO-1 or siRNA negative control. Articular chondrocytes were treated with tert-butyl hydroperoxide (t-BHP) (200 uM; Wako) for 5 h at 24 h after transfection. Apoptotic chondrocytes were quantitated by counting the numbers of cell nuclei stained with Cell Event Caspase-3/7 Green Detection Reagent^TM and NucBlue Live cell stain ReadyProbes^TM (Invitrogen) in three random fields on each duplicate well at a magnification of × 10 under a fluorescence microscope (BZ-9000; Keyence, Osaka, Japan).

Cetuximab (5 mg/mL) was incubated in PBS containing 0.2, 1 or 5% of tert-Butyl hydroperoxide (t-BHP) (Wako) for 3 hr at 37°C. After the incubation, t-BHP was removed from the reaction mixtures by applying the mixtures two times to a PD Spin Trap G-25 column (GE Healthcare) equilibrated with PBS.