Asys uvm340 microplate spectrophotometer

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.

To assess the levels of neurotrophic factors secreted by the cells, the supernatants collected in the sections above were analysed with ELISA kits for brain‐derived neurotrophic factor (BDNF), nerve growth factor (NGF), and glial‐derived neurotrophic factor (GDNF) (RayBiotech, Norcross, GA, USA), according to the manufacturer's instructions. For GDNF and NGF, before performance of the assays, supernatants were concentrated with Amicon Ultra‐4 Centifugal Filter Concentrator units (Merck Millipore), by centrifugation for 40 min at 3100 g, which gave 10‐fold concentration. Each sample was assayed in triplicate, and the final absorbance was determined at 450 nm with an Asys UVM‐340 microplate spectrophotometer (Biochrom). The concentration values of secreted neurotrophic factors were extrapolated from standard curves produced from recombinant proteins provided in the kits.