Elisa development kit

HA levels were determined using a DuoSet enzyme linked immunosorbent assay (ELISA) development kit (R & D Systems).

The nerve growth factor (NGF) assay was performed analogously, as described in Reference [21 (link)]. C6 glioma cell lines were used to measure the NGF of the culture medium containing 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin (PS) in 5% CO₂ incubator. The cells were seeded in a 24-well culture plate (1 × 10⁵ cells/well) and incubated for 24 h. The cells were treated with or without 20 µM of the compounds (1–18), together with serum-free Dulbecco’s modified Eagle’s medium (DMEM) for another 24 h. Released NGF levels from the supernatants from each cell were measured by using an ELISA development kit (R&D System, Minneapolis, MN, USA). Moreover, the cell viability was evaluated by using MTT assay; 6-shogaol used as a positive control, and the results are expressed as percentage of the control group.

The concentration of total TGFβ1 and active TGFβ1 was determined in the L_1–3 IVDs using the ELISA Development kit (R&D Systems) according to the manufacturer’s instructions (n = 3 per group).

The concentration of IL-8, IL-6 and TNF-α in cell culture supernatants was measured with the human Duoset enzyme-linked immunosorbent assay (ELISA) Development kit (R&D System, Minneapolis, MN) according to the manufacturers' instructions.

10 ml of venous blood was drawn into anticoagulant-free tubes between 07:30 and 09:30 after overnight fasting. After 1 h incubation, the serum was separated by centrifugation, aliquoted, and stored at − 70 until analyses the entire sample size could be achieved. The S100B concentration was measured in all samples. Enzyme-linked immunosorbent assay analyses were performed using DuoSet (cat. No DY 1820-05) ELISA Development Kit (R&D System, Minneapolis, MN, USA) according to the manufacturer's instructions, with minor modifications. Plates were coated with the capture antibody (4 µg/ml in PBS) overnight at 4 °C, then washed (3 times) and blocked for three hours in reagent diluent (1% Bovine Serum Albumin (BSA)/Phosphate Buffered Saline (PBS). Serum samples were diluted 1:2 in reagent diluent to avoid any matrix effect. Plates were incubated with 100 μL of samples or standards overnight at RT. All samples and standards were run in duplicates. Detection steps were performed strictly according to the manufacturer’s instructions. All plates were run in one batch, on the same kit lot#, by the same experienced operator. Standard curves ranged from 1500 to 23.4 pg/ml. Intra-assay and inter-assay variability was < 5% and < 10% coefficient of variation (CV) (accordingly).

Left ventricular myocardial concentration of GAL-1 and HIF-1 α and plasma levels of GAL-1 was determined using DuoSet enzyme linked immunosorbent assay (ELISA) Development kit (R&D Systems, Minneapolis, MN, USA) for sandwich ELISA, using standard procedure according to the manufacturer’s instructions. The levels were normalized to total protein concentrations.
Briefly, 96-well plates (Nunc-Immuno Plate MaxiSorp Surface (NUNC Brand Products, A/S, Roskilde, Denmark), were coated with antibody specific for GAL-1 and HIF-1 α. Biotinylated detection antibody and streptavidin conjugated horseradish peroxidase were used for detection of captured GAL-1 and HIF-1 α. Captured GAL-1 and HIF-1 α were visualized using tetramethylbenzidine (TMB)/hydrogen peroxide. Absorbance readings were made at 450 nm, using a 96-well plate spectrophotometer (BioTek ELx800). GAL-1 and HIF-1 α levels in samples were determined by interpolation from a standard curve.

For cAMP assays, confluent cells were grown in 35-mm six-well plates starved overnight by incubation in serum-free α-MEM at 37 °C. The cells were then treated with 100 nmol·L^-1 of human PTH (1–34; Bachem California, Inc.) for 1 h. Cellular cAMP was extracted and concentration measured with the Biotrak enzyme immunoassay system (GE Healthare, Inc., Princeton, HJ). We determined the concentration of total and active TGF-β in the NP tissue using the ELISA Development Kit (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions.