Measurement of secreted TNF and IL-6 by ELISA

TNF and IL-6 levels in the culture medium were measured using commercially available ELISA kits. For TNF, the Mouse TNF alpha ELISA Ready-SET-Go kit (eBioscience) was used. For IL-6, the Mouse IL-6 ELISA MAX Deluxe Set kit (BioLegend) was used. Culture media from LPS-treated or control samples were either diluted or used directly for measurement. All incubation and washing steps were performed according to the manufacturer’s recommended protocols. The optical density values were measured using a FlexStation 3 instrument (Molecular Devices). Cytokine levels were determined by comparison with the standard curves.