Rhmgb1

The recombinant HMGB1 (rHMGB1, Novoprotein, Shanghai, China) series concentrations, ranging from 15.625 ng/ml to 1 μg/ml, and serum diluted with PBS (1∶ 8) from patients or mice, were incubated overnight in 96-well plates at 4°C. Plates were then washed five times in PBS Tween-20 buffer (PBST; 0.05% Tween-20 in PBS). This step was performed after each incubation period. The plates were blocked for two hours at 37°C using 3% bovine serum albumin (BSA) in PBST. After this, rabbit anti-HMGB1 polyclonal antibody (1:1,500, Abcam, U.S.A), diluted in PBS buffer containing 2% skimmed milk, was added to all wells and incubated for two hours at 37°C. The plates were then incubated for one hour at the same temperature, with anti-IgG rabbit antibody conjugated to horseradish peroxidase (1:5,000, Golden Bridge Biotech Co Ltd, Beijing, China). After washing, tetramethylbenzidine (Abcam, U.S.A) was added to each well and plates were incubated in the dark for 10-20 minutes at 37°C. Reactions were monitored by measuring the absorbance at 460 nm (Thermo, U.S.A). All assays were run in duplicate on each test plate.
TNF-α and IL-6 levels were assessed using commercial ELISA kits (4A Biotech Co., Ltd, Beijing, China).

The recombinant HMGB1 (rHMGB1, Novoprotein, Shanghai, China) series concentrations, ranging from 15.625 ng/ml to 1 µg/ml, and serum diluted with PBS (1∶8) from patients or mice, were incubated overnight in 96-well plates at 4 °C. Plates were then washed ve times in PBS Tween-20 buffer (PBST; 0.05% Tween-20 in PBS). This step was performed after each incubation period. The plates were blocked for two hours at 37 °C using 3% bovine serum albumin (BSA) in PBST. After this, rabbit anti-HMGB1 polyclonal antibody (1:1,500, Abcam, U.S.A), diluted in PBS buffer containing 2% skimmed milk, was added to all wells and incubated for two hours at 37 °C. The plates were then incubated for one hour at the same temperature, with anti-IgG rabbit antibody conjugated to horseradish peroxidase (1:5,000, Golden Bridge Biotech Co Ltd, Beijing, China). After washing, tetramethylbenzidine (Abcam, U.S.A) was added to each well and plates were incubated in the dark for 10-20 minutes at 37 °C. Reactions were monitored by measuring the absorbance at 460 nm (Thermo, U.S.A). All assays were run in duplicate on each test plate.
TNF-α and IL-6 levels were assessed using commercial ELISA kits (4A Biotech Co., Ltd, Beijing, China).