Total reactive oxygen species assay kit

The concentration of CCL2 in plasma, as well as the concentrations of IL-1β. IL-6, and TNF-α in the THP-1 cell culture samples were determined using ELISA kits for mice (Abcam, Boston, Massachusetts, USA) following the manufacturer’s protocols. Plasma samples were diluted 1:5 in the binding buffer. Samples were added to wells that had been precoated with immobilized antibodies. Standards were also added to the wells. Next, any unbound biotinylated antibodies were washed away, and horseradish peroxidase (HRP)-conjugated streptavidin was added to the wells. After three more washes, a tetramethylbenzidine (TMB) solution was added to develop the color, which was measured at 450 nm using an ELISA microplate reader (Promega, Madison, Wisconsin, USA). For ROS assays, we used a total reactive oxygen species assay kit from Invitrogen. Samples were prepared and assayed following the manufacturer’s instruction.

Ubenimex were provided by Shenzhen Main Luck Pharmaceuticals, Inc. (Shenzhen, China). LIVE/DEAD^TM Cell Imaging Kit and Total Reactive Oxygen Species (ROS) Assay Kit were purchased from Thermo Fisher Scientific (USA). Cell Counting Kit-8 was purchased from Dojindo (Japan). Annexin V-FITC/PI kit was purchased from BD Biosciences (USA). NAC, 3-MA and Z-VAD-FMK were purchased from Selleck (USA). Primary antibodies: caspase-3 (1:500; catalog # ab13847), parp1 (1:2000; catalog #ab32138), cleaved parp1 (1:2000; catalog #ab32064) and β-actin (1:5000; catalog # ab8226) were purchased from Abcam (UK). ERK (1:1000; catalog # 4695T) and p-ERK (1:1000; catalog # 4370T) were purchased from Cell Signaling Technology (USA). Caspase-9 (1:1000; catalog # 10380-1-AP) was purchased from Proteintech (USA).