Bio plex 5

Concentrations of IFN-γ, IL-1β, IL-6 and TNF-α were simultaneously quantified in plasma using the ProcartaPlex™ immunoassay (eBioscience, San Diego, CA, USA). Cytokine concentrations were determined using analyte specific capture beads coated with target-specific capture antibodies according to the manufacturer’s specifications. The analytes were detected by biotinylated analyte-specific antibodies. Following binding of the fluorescent detection label (SA-PE), the reporter fluorescent signal was measured with the Bio-Plex 200 multiplex suspension array system employing Luminex xMAP technology in combination with the Bio-Plex 5.0 Software (Bio-Rad, Hercules, CA).
Standard curves for each analyte were generated by using the reference analyte concentration supplied and concentrations were calculated using a five-parameter logistic curve-fitting method. Cytokines that were not detected were assigned a value of zero. The sensitivity for the respective cytokines was: IFN-γ: 0.09 pg/mL, IL-1β: 0.14 pg/mL, IL-6: 0.21 pg/mL, TNF-α: 0.39 pg/mL.
Plasma samples of experiment 3.3 were run in duplicate. Since the coefficient of variance for the duplicate samples was small, single samples were run subsequently.

Concentrations of secreted factors were measured using the human magnetic Luminex Assay System according to the manufacturer’s protocol. A Bio-Plex 200 multiplex suspension array system (Bio-Rad, Hercules, CA, USA) was used for signal measurements and detection was analyzed using the Bio-Plex 5.0 Software (Bio-Rad, Hercules, CA, USA). Supernatants of spheroids were taken on day three and seven for subsequent proteomic analysis. Pools of 20 wells each were stored at −80 °C until further use. All samples were measured in duplicates and three biological replicates.

The plasma levels of IL-6, IL-10, IL-12, and IL-18 were measured with a multiplex immunoassay (ProcartaPlex Multiplex Immunoassays, eBioscience, Vienna, Austria). The assay was performed according to the manufacturer's instructions. The fluorescent signal was measured with the Bio-Plex 200 multiplex suspension array system in combination with the Bio-Plex 5.0 Software (Bio-Rad, Hercules, CA, USA). Standard curves were generated with a five-parameter logistic curve-fitting method. Cytokines that were below detection limit were assigned a value of zero. The sensitivities of the assay were 0.9, 0.35, 0.35, and 8.09 pg/ml for IL-6, IL-10, IL-12, and IL-18, respectively.

Cytokine concentrations were determined from the non-pigmented lymph node part, using analyte-specific capture beads coated with target-specific capture antibodies according to the manufacturer’s specifications. The analytes were detected by biotinylated analyte-specific antibodies. Following binding of the fluorescent detection label, the reporter fluorescent signal was measured with the Bio-Plex 200 multiplex suspension array system (Bio-Rad, Hercules, CA, USA) and detected with Bio-Plex 5.0 Software (Bio-Rad, Hercules, CA, USA). The sensitivity for the respective cytokines was as followed: CCL1/I-309 1.73–1260 pg/mL; CCL5/RANTES 8.27–6030 pg/mL; CCL11/Eotaxin 20.23–14,750 pg/mL, CXCL1/GRO alpha 15.30–11,160 pg/mL; IL-8/CXCL8 1.44–1050 pg/mL; CCL2/JE/MCP-1 11.87–8650 pg/mL; CCL8/MCP-2 4.66–3400 pg/mL; CCL17/TARC 30.62–22,320 pg/mL; CXCL10/IP 0.43–310 pg/mL. Analyte levels of complete growth medium mixed with the additives FBS or hPL served as background controls (Supplementary Figure S2).