Human il 10 elisa max standard kit

Gas6, IL-10 and TNF-α levels were measured in supernatants of cell cultures using sandwich ELISA according to standard procedure [32 (link)]. Briefly, 96-well plates were precoated overnight with a capture antibody. Samples from cell culture supernatants were applied to precoated plates in duplicate. Serial dilutions of purified recombinant rhGas6 (R&D Systems) were used to construct a standard curve. Blank wells received serum-free X-Vivo™15 medium. A purified goat polyclonal anti-human Gas6 antibody (R&D Systems) was used for capture. Biotinylated goat polyclonal anti-human Gas6 antibody (R&D Systems), followed by HRP-conjugated streptavidin (Biolegend), was used for detection. The plates were developed with 3,3′,5,5′-tetramethylbenzidine substrate. The reaction was stopped with 2 N sulfuric acid. Absorbance was detected at 450 nm and read with a reference wavelength set at 570 nm using a VersaMAX ELISA microplate reader (Molecular Devices, Sunnyvale, CA, USA). The optical density for each point was the average of duplicate samples. Concentrations were determined using SoftMax software (Molecular Devices) by applying four-parameter logistic regression to the standard curve. IL-10 and TNF-α levels were measured using human IL-10 ELISA MAX Standard kit and TNF-α ELISA MAX Standard kit (Biolegend), following the manufacturer’s instructions.

Human myeloid leukemia HL60 and THP-1 cells (RIKEN Cell Bank, Tsukuba, Japan) were maintained in RPMI1640 medium containing 10% fetal bovine serum, 100 unit/ml penicillin, and 100 µg/ml streptomycin in a humidified atmosphere containing 5% CO₂. Suspensions of cells (10⁵ cells/ml) were cultured with or without test compounds at pharmacological concentrations (30−100 nM) according to our preliminary experiments and the previous reports [10] (link)–[12] (link), [27] (link). Cell numbers were counted in a Z1S Coulter Counter (Beckman Coulter, Fullerton, CA). Cell morphology was examined in cell smears stained with May-Grünwald-Giemsa. NBT reduction was assayed colorimetrically and NBT-reducing activity data were normalized to the cell numbers [28] (link). IL-10 levels in culture media were determined with the Human IL-10 ELISA MAX Standard kit (BioLegend, San Diego, CA).