Tnfα and il 10 elisa kits

The general reagents were purchased from Sigma-Aldrich (UK), unless stated otherwise. RPMI 1640, lipopolysaccharide (LPS), Gentamicin, MTT and NaNO₂, sodium nitroprusside (SNP), penicillin–streptomycin solution, and metformin were procured from Sigma-Aldrich. Recombinant mouse IFNγ cytokine is from eBiosciences (San Diego, CA, USA). CD11b+ human and Mouse MACS Microbeads and LC Columns are from Miltenyi Biotec. Primary antibodies including rabbit polyclonal NOS-2, rabbit polyclonal xIAP, cIAP-1, cIAP-2, LAMP-2, CD-206, rabbit polyclonal β-actin, and mouse monoclonal β-actin are from Santa Cruz biotechnology. Mouse monoclonal arginase-1 is from BD Biosciences. Rabbit monoclonal STAT1 and 3, pp38MAPK, and pNF-kB p65 are from Cell Signalling Technology. Ym-1 antibody was purchased from Stem cell technologies; Fizz-1 antibody was from Abcam. HRP-linked anti-mouse IgG and anti-rabbit IgG are from Cell Signalling Technology. TNFα and IL-10 ELISA kits were purchased from R&D system (Darmstadt, Germany).

Morning fasting venous blood (3 mL) was collected, centrifuged at 4000 r/min for 10 min to separate serum, and stored at −80°C before assay. Human serum IL-17A, TNF-α, and IL-10 ELISA kits were purchased from R&D Systems (Abingdon, UK). Serum IL-17A, TNF-α, and IL-10 levels were detected according to the manufacturer's instructions. Optical density at 450 nm was measured with a spectrophotometer.
SOD and MDA assay kits were purchased from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Serum SOD levels were measured according to the manufacturer's instructions. Briefly, the serum of subjects was mixed with the reagents and incubated for 40 min at 37°C. After the reaction, absorbance at 560 nm was monitored using a spectrophotometer. The samples were then mixed with trichloroacetic acid and incubated for 40 min at 95°C. The absorbance of each sample was measured at 532 nm with a spectrophotometer. MDA concentration was calculated according to the formula provided in the protocol.