Quantikine elisa human tgf β1 immunoassay

To evaluate the production of TGFβ1 by cells in culture, the Quantikine^® ELISA Human TGFβ1 Immunoassay (R&D Systems, Minneapolis, MN, USA) kit was used. To obtain the samples, cells were seeded in 24-well plates. When they reached 80% confluence, medium was switched to 250 µL of phenol red-free DMEM (Thermo Fisher Scientific Inc., HyClone, Rockford, IL, USA) supplemented with 1% FBS and 1% antibiotics. After 24 h, supernatants were harvested, centrifuged at 480 g for 5 min and were stored at −20 °C until ulterior analyses. Before performing the quantification assay, latent TGFβ1 present in the supernatants was activated for its detection by incubating the samples with 1 M of hydrochloric acid, followed by neutralization by adding a solution containing 1.2 M of sodium hydroxide and 0.5 M of HEPES. In parallel, cells remaining in the wells were counted using a TC20^TM automated cell counter (BioRad) to normalize the TGFβ1 measurements.

Extracellular levels of TGF-β1 were assessed by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's protocol (Quantikine ELISA Human TGF-β1 Immunoassay, R&D Systems, Minneapolis, MN, USA). During exposure to 30 μM PQ for 12 days, culture medium was exchanged for new medium every three days, and TGF-β1 levels in conditioned medium (10–12 days with or without PQ exposure) were determined after the removal of cell debris by centrifugation.

Using plasma collected from patients, TGF-β1 was quantified using the Quantikine ELISA Human TGF-β1 Immunoassay (#SB100B) from R&D. For HMGB-1, the human HMGB-1 ELISA Kit (#ST51011) from IBL International (Hamburg, Germany) was used. All assays were performed according to the manufacturer’s instructions. To avoid interassay variations, samples of the same patient were thawed and immediately ran using the same ELISA plate. The optical density was measured at 450 nm in the Multiskan Ascent spectrophotometer (Thermo Scientific, Waltham, MA, USA). Each sample was run in triplicate and the values were averaged.