Maxisorp elisa microplate

The intracellular level of human GDF-15 in THP-1 MΦ was quantified by the DuoSet^® ELISA Development System (R & D Systems, Inc., Abingdon, UK). The capture antibody was coated to a 96-well MaxiSorp-ELISA Microplate (Nunc, San Diego, CA, USA) and incubated overnight at room temperature. According to manufacturers’ instructions, after the blocking step, the samples (2.5 μg protein/well) or standards were added to the well. After incubation with a detection antibody and streptavidin-HRP, we added the substrate solution (SigmaFast™ OPD, Sigma-Aldrich Chemie GmbH) to each well and there incubated for 30 min in the dark. The reaction was stopped with 50 μL 3 M HCl. The GDF-15-protein level (pg/mL) was measured with an ELISA reader (Tecan Deutschland GmbH, Crailsheim, Germany) at OD_490/655nm.

The release of IL-6, IL-10, and TNF-α was quantified using an enzyme-linked immunosorbent assay (ELISA). According to the manufacturer's instructions, cytokines were determined in the culture medium using the assay DuoSet ELISA™ Development kit (R&D Systems Europe, Ltd., Abingdon, UK) (Table 4). The Capture Antibody was coated to a 96-well MaxiSorp™-ELISA Microplate (Nunc, San Diego, USA) and incubated overnight at room temperature. After the blocking, 100 µl samples or standards were added to the well. After the incubation with the detection antibody and streptavidin-horseradish peroxidase (HRP), the substrate solution [SigmaFast™ OPD (o-Phenylendiamin-dihydrochlorid), Sigma-Aldrich Chemie GmbH] was added to each well and incubated for 30 min in the dark. The reaction was stopped with 50 μl 3 M HCl and the optical density (OD) was measured at 490 nm and reference at 655 nm (OD 490/655) using a Sunrise microplate ELISA reader (Tecan Deutschland GmbH). The concentration of cytokines released into the medium was calculated by interpolation from the respective standard curves and normalized against the protein concentration.