Human elisa duoset

Human PASMC were plated in 96-well plates (5000 cells/well) and cultured in DMEM/15 % FCS until 90 % confluent; cells were serum-starved overnight and then tumour necrosis factor (TNF)-α (10 ng/ml) for 24 h used as the stimulus in the presence or absence of dexamethasone (10⁻⁸–10⁻⁶ M) or an IKK-2 inhibitor (AS602868, 0–3 μM). After 24 h, the supernatant was aspirated and stored at −20 °C and MTT viability assays (Sigma Aldrich) were performed. ELISA measurement of IL-6 and CXCL8 levels (Human ELISA DuoSet, R&D Systems) were performed on supernatants according to the manufacturer’s instructions as follows using distinct capture and detection antibodies: 100 μl of assay diluent, 50 μl of standard or cell culture supernatants were pipetted to each well sequentially, and incubated at room temperature for 2 h. After incubation, wells were washed 4 times and incubated with 100 μl of conjugate for 1 h. Wells were then washed 4 times and incubated with 200 μl of substrate solution for 30 min. The plates were read at 450 nm using a BioTek plate reader immediately after 50 μl of stop solution was pipetted to each well.

Anthropometrics and fasting blood samples were collected after an overnight fast. All participants were asked not to engage in excessive physical exercise or alcohol intake the day before or in the morning of clinical investigations. Venous blood samples were collected and serum frozen at −80°C. The homeostasis model assessment insulin resistance index (HOMA-IR) was calculated using the formula of serum fasting insulin (μU/mL) ∗ fasting glucose (mmol/L)/22.5 [29 (link), 30 (link)]. Soluble CD163 was quantified in serum samples using an in-house enzyme-linked immunosorbent assay, as previously described [31 (link)]. IL-6 was measured with a high sensitivity ELISA (R&D Systems, USA) and MCP-1 was measured with a human ELISA DuoSet (R&D systems). sCD163, MCP-1, and IL-6 were measured and described in a previous study [23 (link)].