Human il 6 elisa kit

Intracellular IL-6 was measured using a Human IL-6 ELISA Kit (RAB0307; Sigma-Aldrich) following the manufacturer’s protocol. The total cells were lysed and diluted to a protein concentration of 1 mg/mL with indicated diluent buffer before measurement.

Plasma concentrations of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were determined by using ELISA kits: Human IL-6 ELISA Kit, RAB0306-1KT, LOT#: 0428I140; Human Tumor Necrosis Factor alpha ELISA Kit, RAB0476-1LT, Lot#: 0422I193 (Sigma-Aldrich, St. Louis, MO, USA). Brifly, calibration curves were prepared by using protein standards with suitable serial dilutions. Fifty microliters of plasma were diluted with buffer solution two times for sample incubation. After adding antibodies and reporter reagent for a specified incubation time, the signals were determined with a microplate spetrophotometer (450 nm). The optical density (OD) values were used to calculate the analyte concentrations. Urine creatinine concentrations were also determined by using a commercially available ELISA kit: Parameter^TM Creatinine Assay, KGE005, LOT#: P289536 (R&D Systems, Minneapolis, MN, USA). Ten microliters of urine were diluted with buffer solution 20 times for sample incubation. After adding alkaline picrate solution for a 30 min incubation time, the signals were determined with a microplate spetrophotometer (490 nm).

Human myotubes grown in 6-well EMC coated Costar plates (Corning^®) were stimulated via carbon electrodes (C-dish, Ionoptics, Ireland) by applying continuous, low-frequency electrical pulse stimulation (EPS) (single, bipolar pulses of 2 m, 30 V, 1 Hz) for 24 h from day 6–7 of the differentiation period, as described previously (Nikolic et al., 2012 (link)). Electrical pulses were generated by a self-designed pulse generator (the Electronics Lab, Institute of Chemistry, University of Oslo, Norway), and the effect was investigated by measuring IL-6 concentration in the supernatants before and after EPS. IL-6 was measured spectrophotometrically by sandwich ELISA according to the producer’s protocol (Human IL-6 ELISA kit, SigmaAldrich), and the concentration calculated from a standard curve.

PGE2 and MMP released into conditioned media were quantified using a commercially available enzyme immunoassay kit (R&D Biosystem) as previously described [29 (link)]. For IL-6, we proceeded in the same way but using the Human beta-NGF ELISA Kit and the Human IL-6 ELISA kit (Sigma Aldrich). The immunoassays were all carried out in accordance with the manufacturer’s protocol. Absorbance was determined at 450 nm with a wavelength correction set at 540 nm using the Multiskan GO spectrophotometer (Thermo Scientific).

The HL‐60 cell line (RBRC‐RCB0041) was provided by RIKEN BRC through the National Bio‐Resource Project of MEXT, Japan. HL‐60 cell culture and neutrophilic differentiation were previously described (Shuto et al, 2007). Neutrophilic differentiation was induced by exposing HL‐60 cells to 1.3% DMSO for 5 day. At 3 day after exposure to 1.3% DMSO, HL‐60 cells were transfected with control or miR‐223 AS ODN using Lipofectamine 3000 (Life Technologies) in Opti‐MEM with 1.3% DMSO (Life Technologies). On 2 day after transfection, cells and conditioned media were harvested and applied to gene expression assays and ELISA (Human IL‐6 ELISA Kit, Sigma‐Aldrich, St. Louis, MO, USA).