Human il 6 elisa kit

Cells were cultured at 5 x 10⁴ cells/mL in DMEM supplemented with 1% D-FBS and ITH for 2 days. The CMs were collected by centrifugation and the amounts of IL-6, IL-6sR, and CXCL1 were determined using a human IL-6 ELISA kit (Thermo Scientific), a human IL-6sR Quantikine (R&D Systems) and a human CXCL1 Quantikine (R&D Systems). The culture supernatants were also applied onto human cytokine antibody array (C series 2000; RayBio). The amounts of PGE2, 6-keto PG, thromboxian B₂, and leukotriene B4 were determined using quantitative kits from Cayman.

T98G (ATCC® CRL1690™) cells were grown in 12‐well plates (1 × 10⁵ cells per well) for knockdown experiments or 24‐well plates (5 × 10⁴ cells per well) in DMEM high glucose, supplemented with 10% FCS, sodium pyruvate (1 × 10⁻³m), penicillin (100 U mL⁻¹), and streptomycin (100 µg mL⁻¹) at 37 °C and 5% CO₂ for 24 h. Before incubation with LPS and test compounds, the medium was changed to DMEM supplemented with 1% charcoal‐stripped FCS, sodium pyruvate (1 × 10⁻³m), penicillin (100 U mL⁻¹), and streptomycin (100 µg mL⁻¹) for 12 h, or Nurr1 knockdown was performed by transient transfection for 24 h as outlined above. The cells were then treated with LPS (1 µg mL⁻¹) to induce inflammation and simultaneously incubated with simvastatin (10 × 10⁻⁶m), fluvastatin (30 × 10⁻⁶m) or pravastatin (30 × 10⁻⁶m), and 0.1% DMSO, or 0.1% DMSO alone as untreated control. Each sample was repeated independently at least three times. Following overnight (12 h for knockdown or 24 h) incubation, 100 µL of the respective supernatants was collected and assayed for IL‐6 using the Human IL‐6 ELISA Kit (Cat# KHC0061, Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Absorbance at 450 nm was measured with a Spark 10M luminometer (Tecan Group AG).

IL-6 expression by ME-180 and peripheral blood mono-nuclear cells (PBMCs) in response to HSV-2 infection or GpC stimulation was quantified by ELISA. In brief, PBMCs were isolated from single buffy coats of healthy donors obtained from NHS Blood and Transplant using Histopaque (Sigma-Aldrich). ME-180 cells with or without TLR9 overexpression and PBMCs were either infected with HSV-2 or stimulated with CpG or control GpC for 24 h. Cell culture supernatants were collected and IL-6 was quantified by ELISA using human IL-6 ELISA kit (Thermo Fisher Scientific) according to the manufacturer's instructions.

The frozen human peripheral blood mononuclear cells were obtained from Zen-Bio Inc. (Research Triangle Park, NC, USA). As the European Pharmacopoeia’s guideline recommendation for a cellular test investigating cytokine output (the monocyte-activation test (2.6.30) published in the European Pharmacopoeia Supplement 9.2), each PBMC pools (male and female PBMCs) was prepared from four healthy donors, in order to reduce the effects of donor variability. Healthy donors were matched for race (Caucasian) and age (31±3). Each cryovial contained similar content of lymphocytes and monocytes, as reported in the furnished data sheets (lymphocytes = 89.35% ± 1.91; monocytes = 10,65% ± 1.91).
Cell culture media and foetal bovine serum were obtained from Cambrex Bio Science Walkersville, Inc. (Walkersville, MD). Cortisol (HC, hydrocortisone ≥98% (HPLC), powder) was obtained from SIGMA Italia (Milan, Italy). The Human IL-6 ELISA Kit, Human IL-10 ELISA Kit and Human IL-8 ELISA Kit were obtained from Thermo Fisher Scientific Inc. (Rockford, USA). The Human IL-4 ELISA Kit was obtained from Cloud Clone Corp (Katy, USA). The Human KYN ELISA Kit was obtained from Cusabio Biotech Co (Baltimore Avenue, College Park, USA). All other reagents were from standard commercial sources.

IL-6 levels were assessed in triplicate samples using the Human IL-6 ELISA Kit (Thermo Scientific) according to manufacturer’s instructions. Absorbance was determined at 450±10 nm with a microtiter plate reader (Bio-Tek Instruments).