Human chemokine array kit

Conditioned media of macrophages stimulated for 18 h with β-glucans were tested for the presence of cytokines using human IL-6, IL-10 and TNF-α ELISAs (Biolegend, San Diego, USA) according to the manufacturer's instructions. Conditioned media of M(IL-4) stimulated with curdlan, zymosan or yeast-b were also tested for the presence of chemo-attractants using a Human Chemokine Array kit (ARY017, R&D Systems).

In total, 200 μg of protein was extracted from cell lysates from MDAMB231 cells to detect the expression of 31 chemokines using the human chemokine array kit (#ARY017, R&D Systems). Images of membranes were acquired with ChemiDoc (BioRad) and dots were quantified using QuickSpots (Western vision) software. At the same time point, supernatants of MDAMB231 and HCC1395 cells were collected to detect MCP-1 levels with the human CCL2/MCP-1 Immunoassay (#SCP00, R&D Systems).
See Supplementary Methods for details regarding RNA-seq analysis.

The Human Chemokine Array Kit (R&D Systems, Abingdon, UK) was used to measure chemokine concentration in supernatants of HUVECs treated for 24 h with 10 μM S1P or left untreated. CXCL8 release by HUVECs treated with 10 µM S1P, 10 μM CYM5442, 10 μM CYM5541 or 10 μM FTY720P at sequential timepoints or left untreated was measured by an ELISA assay (PeproTech, Cheshire, UK).

After co-culture of 3DJPCs/3DOBJPCs and M1/M2 macrophages for five days, supernatants from macrophage chambers were collected and stored at −80 °C. The human chemokine array kit (R&D Systems, Minneapolis, MN, USA) was used to analyze semi-quantitative secretion levels in collected supernatants. Briefly, chemokine array membranes were pretreated with a blocking buffer for one hour at room temperature and then incubated overnight at 4 °C with the mixtures of sample supernatant and antibody cocktail. After washing, membranes were incubated with diluted streptavidin-HRP for 30 min at room temperature. After additional washing, membranes were incubated with 1 mL of chemiluminescent reagent mixture and exposed to radiographic film (Cytiva, Marlborough, MA, USA) for 10 min. After scanning the developed and fixed X-ray film, positive signals were determined semi-quantitatively using the ImageJ 1.54g Java 1.8.0_345 64-bi software (NIH, Bethesda, MD, USA) and the pixel density ratio of target protein/spots to reference spots was calculated for data analysis.

Cytokine and chemokine levels were analyzed in CM from CAF-173 treated with OSM (30 ng/mL) or vehicle for 72 hours (n = 4), and from MDA-MB-231-hOSM and corresponding control cells (n = 6). A panel of 31 human chemokines was analyzed by Human Chemokine Array Kit (Proteome Profiler Array, R&D Systems), and VEGF levels were quantified by Human VEGF Quantikine ELISA Kit (R&D Systems) following the manufacturer’s instructions. Mouse VEGF, CXCL1, and CXCL16 levels in plasma from 14-week-old MMTV-PyMTOsmr-KO, -HET, and -WT mice were analyzed by mouse Premixed Multi-Analyte Kit (Magnetic Luminex Assay, R&D Systems) following the manufacturer’s instructions. Detection was carried out with the MAGPIX detector and data analysis was performed using xPOTENT software, both from R&D Systems.