Quantifying Cytokine Levels in Cell Cultures

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.