Bio plex express assay

The cytokines, IL-6, IL-8, and TNF-α, were measured using a custom-made multiplex assay (Bio-Plex Express Assay, Bio-Rad Laboratories, Hemel Hempstead, UK) based on Luminex xMAP technology according to the manufacturer’s instructions. Briefly, the standard was reconstituted and diluted in a fourfold dilution series. Antibody coupled capture beads were prepared and plated. After washing using a Bio-Plex Pro™ wash station (Biorad), diluted samples and standards were added to the beads in the wells. The plate was incubated on a shaker and after incubation and wash, detection antibodies were added to each well and after the streptavidin-phycoerythin solution (R&D Systems, Abingdon, UK) was added to the wells. In the last incubation step, beads were resuspended in assay buffer and the plate was read with a BioPlex 200 instrument equipped with BioManager analysis software (BioRad). The absolute concentrations of the samples were determined by comparing the bead colour and mean fluorescence intensity from each set of beads against an automatically optimized and manually verified standard curve. The cytokine concentration was presented as pg/mL.

The cytokine expressions were measured using a combination of a custom-made premixed x-Plex assay for detection of TNF-α, IFN- γ, IL-6, IL-8, IL-12p70, IL-17, MCP-1, and IL-1R, and a manually added single plex set for detection of IL-12p40 (Bio-Plex Express Assay, Bio-Rad Laboratories, Hemel Hempstead, UK), using Luminex xMAP technology according to the manufacturer’s instructions.
Briefly, all samples were incubated with sets of distinctly color-coded beads conjugated with capture antibodies directed against a specific analyte. After washing, a biotinylated detection antibody was added and allowed to react with the bound proteins. After another washing step, streptavidin conjugated to the fluorescent indicator phycoerythrin was added. Then, the final washing step was followed by acquisition of data using a BioPlex 200 instrument equipped with BioManager analysis software (BioRad). The absolute concentrations of the cytokines were determined by comparing the bead color and mean fluorescence intensity from each set of beads against an automatically optimized and manually verified standard curve. The cytokine concentration was presented as pg/mL.

Supernatants from HuALN^® reactors were collected over a period of 28 days. Since (re-) stimulation took place after the collection of supernatants, changes in cytokine secretion can be detected one day later, at the earliest. Supernatants were analyzed with a multiplexed suspension array system (Bio-Plex 200, Bio-Rad, Munich, Germany). A Bio-Plex Express assay (Bio-Rad) for six custom analytes was used to quantify the cytokines IL-2, IL-4, IL-10, GM-CSF, IFN-γ, and TNF-α. The assay was performed according to the manufacturer’s protocol, but conducted fully-automated on a Tecan Freedom Evo 200 (Tecan, Männedorf, Switzerland) with an in-house-developed Tecan Freedom EVOware^® script in order to lower the variance from manual handling [27 (link)]. All samples were tested in duplicate. The results were analyzed with Bio-Plex Manager 6.1 (Bio-Rad, Munich, Germany) using the logistic five-point regression method.