Human il 6 duoset elisa kit

Human embryonic kidney cell line HEK293 and human lung carcinoma cell line A549 were purchased from American Type Culture Collection (USA) and cultured in Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich, St Louis, MO, USA) supplemented with 10% (v/v) heat-inactivated bovine calf serum (BCS; Thermo Fisher Scientific, Waltham, MA, USA) and 100 units/mL penicillin and streptomycin (Invitrogen, Calsbad, CA) at 37 °C in a humidified atmosphere of 5% (v/v) CO₂. The Expi293F cells (Thermo Fisher Scientific, Waltham, MA, USA) were cultured in Expi293 Expression Medium at 37 °C in a humidified atmosphere of 8% CO₂. 2,2′-Azinobis[3-ethylbenzothiazoline-6-sulfonic acid] (ABTS) and 30% hydrogen peroxide were from Sigma-Aldrich. Horseradish peroxidase (HRP)-conjugated goat anti-human or -mouse IgG Fc and HRP-conjugated goat anti-mouse IgG F(ab′)₂ secondary antibodies were from Jackson Immunoresearch Laboratories (West Grove, PA). Recombinant Interleukin (IL)-1β, IL-6 and human IL-6 DuoSet ELISA kit were purchased from R&D Systems (Minneapolis, MN, USA). Recombinant MMP-9 and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Cells were plated at 50,000 cells per well in 24-well culture plates in DMEM supplemented with 10% FBS and penicillin/streptomycin and allowed to attach overnight. The next day, cells were gently rinsed with sterile PBS and media was replaced with 500 μL DMEM (without FBS or penicillin-streptomycin). After 72 hours of incubation, conditioned media were removed and centrifuged for 10 min at 10,000 RCF at 4°C. Supernatants were transferred to fresh 1.7-mL microcentrifuge tubes and frozen at -80°C for subsequent analysis. The concentration of IL-6 in the cell culture supernatants was determined using the Human IL-6 DuoSet ELISA kit (R&D Systems DY206) according to the manufacturer’s instructions. Plates were read at 450 nm on the Biotek Epoch Microplate Spectrophotometer, with wavelength correction at 540 nm. Assays were performed in technical duplicate. The number of biological replicates is indicated in the respective figure legends. GraphPad Prism was used to conduct Student’s two-tailed t-test to determine whether changes in concentration of secreted IL-6 were statistically significant between parental PE/CA-PJ49 cells and PE/CA-PJ49 cells that had acquired resistance to cetuximab.

The determination of IL1ß and IL6 protein from cell culture supernatants was performed with commercially available sandwich enzyme-linked immunosorbent assays (human IL-1 beta/IL-1F2 and human IL-6 DuoSet ELISA Kit, R&D systems, Wiesbaden, Germany) according to the manufacturer’s instructions. For all determinations, 100 µL of 1:5 diluted culture supernatant was used, and determination was performed in technical duplicates.