Manager 6

Serum levels of CCL23 and IL-4 were measured by multiplex immunoassay according to the manufacture's instruction using a Bio-Plex 200 system and Bio-Plex Manager 6.0 software (Bio-Plex Pro Human Chemokine Panel, #171AK99MR2, Bio Rad, Hercules, CA, USA).

In a subset of 45 ESCC patients, the serum concentration of 48 cytokines, chemokines, and growth factors was quantified using the BioPlex 200 platform (Bio-Rad, Herkules, CA, USA), incorporating Luminex xMAP^® technology. This flow cytometry-based method allows for the simultaneous quantification of multiple analytes in real-time. It utilizes magnetic microspheres conjugated with monoclonal antibodies and fluorescent reading. Two Bio-Plex Pro™ Human Cytokine, Chemokine, and Growth Factor Magnetic Bead–Based Assays—Panel I (27-plex) and Panel II (21-plex)—were used. The 27-plex included the following analytes: eotaxin, IL-1β, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12p70, IL-13, IL-15, IL-17, IFNγ, IP-10, FGF-2, G-CSF, GM-CSF, MCP-1, MIP-1α, MIP-1β, PDGF-BB, RANTES, TNFα, and VEGF-A. The 21-plex included: IL-1α, IL-2Rα, IL-3, IL-12p40, IL-16, IL-18, CTACK, GRO-α, HGF, IFN-α2, LIF, MCP-3, M-CSF, MIF, MIG, β-NGF, SCF, SCGF-β, SDF-1α, TNF-β, and TRAIL. The concentration of IL-1α, IL-12p40, MCP-3, M-CSF, and TNF-β was below the limit of detection in most of the samples, and they were, therefore, excluded from analysis. All analyses were conducted in duplicates and following the manufacturer’s instructions. Standard curves were drawn using 5-PL logistic regression, and the data were analyzed using BioPlex Manager 6.0 software.

A Bio-Plex Pro test kit was used to measure the mice’s serum concentrations of a number of cytokines (Bio-Rad Laboratories). The assay plate received a total of 50 μl of antibody-conjugated beads. 50 μl of diluted samples, the blank, standards, and the controls were applied to the plate after the samples had been diluted by 1:4. The plate was shaken vigorously at 300 rpm for 30 minutes while being incubated at room temperature (RT) in the dark. The plate received a total of 25 μl biotinylated antibody after three washes with 100 μl wash buffer. The plate was shaken vigorously at 300 rpm for 30 minutes in the dark at RT with 100 μl washing buffer 3 times. The plate was then filled with a total of 50 μl streptavidin-phycoerythrin (PE) and incubated for 10 minutes in the dark at RT with 300 rpm shaking. A Bio-Plex protein array reader evaluated the samples after three washes. Data collection and assay analysis were done using Bio-Plex Manager 6.0 software.

Measurement of 94 serum markers was performed using in house developed and validated multiplex immunoassay (laboratory of translational immunology, University Medical Center Utrecht) based on xMAP technology (Luminex Austin TX USA) [25 (link), 27 (link)]. An overview of the markers is shown in Table 2 (further detail in S1 Table). For the measurement of biomarkers naturally occurring in high concentrations, samples were diluted either 1:100 (Adipsin, Cathepsin S, Cathepsin L, Chemerin, Leptin, PAI-1, CCL18, RBP-4, Resistin, SAA-1, TIMP-1, TPO, sCD14, sICAM and sVCAM) or 1:1000 (Adiponectin). Acquistion was performed with a Biorad FlexMap3D system using Xponent 4.2 software. Serum values were calculated using Bio-Plex Manager 6.1.1.

Cytokine (CXCL1, CXCL2, IL‐6, CXCL8, CCL2, and TNF‐α) quantification was performed using a customized all in one Bio‐Plex Pro Human Chemokine 6plx EXP kit (17002259, Bio‐Rad). The cytokines in the collected supernatants were measured by the Luminex‐based multiplex technique according to the manufacturer's instructions (Bio‐Rad). The concentrations were calculated with Bio‐Plex Manager 6.0 by comparison with the standard curves. The detection sensitivity ranged between 1 pg and 40 μg of protein per 1 ml.