Opteia human il 8 elisa kit

HEK293A cell culture supernatants were collected after 20hstimulation, and the IL-8 concentration was determined by a capture enzyme-linked immunosorbent assay (ELISA) using the OptEIA Human IL-8 ELISA Kit (BD Biosciences), in accordance with the manufacturer's instructions, except that signal was detected using a chemiluminescent substrate (SuperSignal West Pico, Pierce). Recombinant human IL-8 provided in the kit was used to generate standard curves.

The measurement of IL-8 response in 8 days post-plating HT-29 cells by bacterial cells, spent medium and OMVs was carried out as previously described (Kainulainen et al., 2013 (link)). In brief, the bacterial suspensions were washed with McCoy 5A medium supplemented with 10% FBS and adjusted to OD_600nm 0.25. Bacteria were diluted to 1:10, 1:100, and 1:1,000 and incubated on the HT-29 cells for 3 h at 37°C in a CO₂ incubator. Spent media from broth cultures of O. splanchnicus 57, B. fragilis type strain and E. coli K12 were prepared by adjusting the bacterial culture to OD_600nm 1.0, pelleting the bacteria by centrifugation and filtering the supernatant through 0.2 μm filter to remove any remaining cells. Spent medium preparation was diluted to 1:4 and 1:2 using McCoy 5A medium with supplements. Bacterial growth medium (GAM) diluted with McCoy 5A medium was used as control. OMVs were diluted to McCoy 5A with supplements and added on monolayers using 10¹⁰, 10⁹, 10⁸, and 10⁷ concentrations. Three technical replicates (parallel wells) were used in each experiment. OptEIA Human IL-8 ELISA kit (BD Biosciences, United States) was used according to the manufacturer’s instructions to measure the concentration of the chemokine in the culture media.

Cell debris was removed from the culture media by centrifugation; then, supernatant IL-8 concentrations were measured using an OptEIA Human IL-8 ELISA Kit (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer’s instructions. Briefly, the ELISA plates were coated with capture antibody overnight at 4 °C, washed with PBS containing Tween-20, blocked overnight with PBS supplemented with 10% (v/v) fetal bovine serum at 4 °C, and then incubated with serial dilutions of the samples and IL-8 standards. Following treatment with detection antibody and tetramethylbenzidine substrate, the absorbance was measured at 405 nm using an ELISA reader. The assay detection limit was 3.1 pg/mL IL-8.

Cells of interest were seeded in 96-well plates. Next day, the medium was replaced with fresh medium containing the reagents or stimulator cells of interest. After an additional day, supernatants were analyzed for IL8 expression using the BD OptEIA^TM human IL8-ELISA kit (BD Biosciences, NJ, USA).

CD40-responsive cells (HT1080-CD40 or U2OS) were seeded in 96-well plates (2 × 10⁴ cells per well) and grown overnight. The next day, medium was changed to minimize the background of constitutive IL8 production, and cells were stimulated overnight with the anti-CD40 constructs as indicated. For cocultures CD40-responsive cells were supplemented with a similar number of HEK293 cells transfected with empty vector or expression vector encoding memCD40L or FcγRIIB along with the different antibody fusion proteins. The amount of IL8 in the supernatant was determined using the BD OptEIA^TM human IL8-ELISA kit (BD Biosciences, NJ, USA) according to the manufacturer’s protocol. Cocultures with memCD40L expressing transfectants served to define the maximal CD40-induced IL8 response.