Recombinant proteins

We set out to optimize the initial biosensor chip configuration by comparison to an established Luminex assay. First, we performed a dose response curve examining the limit of detection for the 11 analytes that were evaluated in both the GCFP assay and the Luminex data using parameters that had been employed with the first-generation GCFP chip. Chips were spotted as described above with the capture antibodies for the following analytes: TNF-α, IL-7, IL-6, IL-4, IL-21, IL-2, IL-1β, IL-17A, IFN-δ, CCL-3, CCL20. Recombinant proteins (R&D Systems, MN, USA; Shenandoah Biotechnology, Inc, PA, United States) were then diluted to 5 ng/mL, 1 ng/mL, 200 pg/mL, and 1 pg/mL and recirculated over the chip as described above. Detection ratios were calculated, and the limit of detection was determined for each matched pair set by GCFP. We then optimized the capture and secondary antibodies to reach values detectable with the Luminex MIA assay.

Cell culture media were purchased from Life Technologies (Grand Island, NY). FBS was from Atlanta Biologicals (Flowery Branch, Georgia). Recombinant proteins and neutralizing antibodies were purchased from R&D systems (Minneapolis, Minnesota). Phycoerythrin (PE)-conjugated anti-F4/80 (T45-2342) and allophycocyanin (APC)-conjugated anti-CD11b (M1/70) antibodies were from BD Biosciences (San Jose, California). PE-conjugated anti-c-Met (eBioclone 7) antibody was from eBioscience (San Diego, California). CD31 antibody was from Abcam (Cambridge, Massachusetts). Antibodies used in western blot were purchased from Cell Signaling Technology (Danvers, Massachusetts). Inhibitors were purchased from Selleckchem (Houston, Texas).

Materials were purchased from SIGMA-Aldrich (Poole, UK) unless otherwise stated. Oligonucleotide production and DNA sequencing services were from MWG-Biotech (Ebersberg, Germany). HPLC materials were from Cytiva (Amersham, UK). Recombinant proteins and the small-molecule CXCR6 antagonist ML-339 were from R&D Systems (Bio-Techne Ltd., Abingdon, UK). Heparin dp8 and heparan sulfate (HS) used in the bio-layer interferometry (BLI) assays were from Iduron (Alderley Edge, UK). Primary antibodies used in Western blotting were sheep anti-human CXCL17 pAb (#AF4207), mouse anti-CXCL17 mAb (#MAB4207), and mouse anti-human CXCL4 mAb (#MAB7951) all from Bio-Techne. These were detected with either protein G-conjugated HRP or goat anti-mouse IgG Alexa Fluor Plus 800n, both from Thermofisher (Paisley, UK). All flow cytometry antibodies were purchased from BioLegend unless otherwise stated.