Human il 8 elisa kit

HepG2 cells were diluted to a concentration of $5\times {10}^5$ cells/mL and plated with 1  $\mathrm{m}$ L in 12-well plates. The cells were incubated at 37°C with 5% CO₂ for 1 h to attach to the bottom of the wells. Kaempferol at 40  $\mu$ g/mL and prescribed concentrations of 10^–6 M or 10^–7 M dexamethasone (Sigma Aldrich, St. Louis, MO), were added in volumes of 500  $\mu$ L. After pre-treatment for 48 h, 5 ng/mL of IL-1β was added to pre-treatment groups for stimulation. The cells were incubated at 37°C with 5% CO₂ for 6 h and the supernatants were subsequently collected for IL-8 quantification using a human IL-8 ELISA kit (BD Biosciences). The dilution of IL-8 measurement was 1:1. All experiments were replicated at least three times with duplicate ELISA measurements of each sample.

After cell treatment, condition medium was collected and centrifuged. Supernatant was saved and properly diluted with PBS containing 10% FBS. IL-8 protein concentration in condition medium was determined by using a human IL-8 ELISA kit (BD Biosciences, San Diego, CA, USA).

HT1080 cells were seeded in 96-well cell culture plates (Sarstedt, Germany, #83.3924) (2 x 10⁴ cells/well) and the next day cells were challenged with reagents of interest for an additional 24 h. As a positive control, cells were stimulated with anti-Flag antibody M2 oligomerized Flag-TWEAK which mimics the activity of membrane TWEAK (17 (link)). Cell culture supernatants were collected and analyzed with respect to their IL8 content using the human IL8 ELISA Kit (BD Biosciences, Heidelberg, Germany, #555244).

Human IL-8 ELISA kit (BD Biosciences, Cat. No. 555244) was used to quantify IL-8 in culture supernatants according to the manufacturer suggested protocol. Briefly 100 μL diluted capture antibody was added to each well of 96-well plates and incubated overnight at 4 °C. The following day, the unbound capture antibody was washed thrice with 300 μL 1X wash buffer (supplied with the kit) for 5 min each. The wells were blocked with 200 μL assay diluent for 1 hr at room temperature followed by three washes each time with 300 μL 1X washing buffer for 5 min each. Next 100 μL standard or samples were added to each well and the plate was incubated for 2 hr at RT followed by 5 times wash with 300 μL 1X washing buffer. 100 μL working 1X detection solution (Detection Ab+SAv-HRP at 1:250 dil) was added to each well and the plate was incubated for 1 hr at RT followed by 7 washes with 300 μL 1X washing buffer. Finally, 100 μL substrate solution was added to each well and incubated 30 mins at RT in dark. In order to stop the reaction, 50 μl stop solution was added to each well. All samples were set as duplicates and the absorbance was measured at 450 nm using a microplate reader (BioTek, EL800 Systems).

The production of IL-8 was determined in the conditioned media of the induced-macrophages using the Human IL-8 ELISA Kit (BD Biosciences, San Diego, CA, USA), and following the manufacturer’s instructions.