Elisa max standard set human il 10

IL-10 levels in cell culture supernatants were measured using the ELISA MAX™ Standard Set Human IL-10 (BioLegend) or Human IL-10 ELISA Set (Diaclone), according to the manufacturer’s instructions, using ELISA Coating Buffer (BioLegend). IL-13 in cell culture supernatants was measured using the Human IL-13 ELISA Development Kit (HRP) (Mabtech) or Human IL-13 DuoSet ELISA (R&D Systems) according to the manufacturer’s instructions. For IL-17A measurement Human IL-17A ELISA Development Kit (HRP) (Mabtech) was used, according to the manufacturer’s instructions. Nunc-Immuno™ MicroWell™ 96 well plates (Sigma-Aldrich) were used. Plates were developed using the TMB Substrate Set (BioLegend), with H₂SO₄ added to stop the reaction. Absorbance was read at 450 nm and 570 nm wavelengths using an Epoch 2 Microplate Spectrophotometer (BioTek) or an Asys UVM340 microplate spectrophotometer (Biochrom). The absorbance at 570 nm was subtracted from that at 450 nm, and the concentrations were calculated based on standard curve equations.

Secreted IL-10 or IFN-γ in the culture medium was measured via ELISA (ELISA MAX Standard Set Human IL-10, or ELISA MAX Deluxe Set Human IFN-γ, BioLegend, Koblenz, Germany), according to the manufacturer’s instructions.

IL-10 and IL-17 secretion was measured in culture supernatants of B cells by ELISA (ELISA Max Standard Set Human IL-10, Biolegend and Human IL-17A ELISA Ready-SET-Go!, eBiosciences, San Diego, CA, USA), according to the manufacturer’s instructions. The supernatants were collected after 43 h of culture and before PIB addition and stored at −80°C until the ELISA assay was performed.