U plex biomarker group 1 mouse assay

Excised tumors were resuspended in T-Per Tissue Protein Extraction Reagent (ThermoFisher Scientific) and homogenized using 2.8 mm ceramic (zirconium oxide) beads (Precellys Lysing kit) and Precellys Evolution Homogenizer. Tumor lysates were recovered after spin at 13 000 rpm for 15 min at 4°C and analyzed for cytokine levels using U-PLEX Biomarker group 1 mouse Assay (35 analytes; Meso Scale Diagnostics) and ELISAs specific for the mouse chemokines XCL1 (ThermoFisher Scientific) and CXCL9 (ThermoFisher Scientific), following manufacturers instructions.

Bone homogenates stored at −80°C were thawed, centrifuged (9000 g, 4°C, 10 min) and the supernatants collected for cytokine and chemokine quantification. Those were measured using a Milliplex MAP Mouse cytokine/chemokine Magnetic Bead Panel (Merck) for: G-CSF, IFN-γ, IL-2, IL-4, IL-6, IL-10, IL-13, IL-17A, KC, MCP-1 and TNF-α. Additionally, spleen single cell suspensions resuspended in cRPMI were plated in 24-well plates (1×10⁶ cells/well) and were stimulated with UV-killed S. epidermidis (at two, ten and 50 bacterial cells/splenocyte) and then incubated at 37°C, 5% CO₂. Culture supernatants were collected 48 h later. Using Milliplex MAP Mouse cytokine/chemokine Magnetic Bead Panel (Merck) the following analytes were measured: IFN-γ, IL-2, IL-4, IL-10, IL-17A and TNF-α. Bone homogenates from the IL-17A study were centrifuged (9000 g, 4°C, 10 min) and supernatants were collected and stored at −80°C until use. IFN-γ, IL-4, IL-6, IL-17A, IL-17F, IL-22, IL-33 and KC were then measured in those samples using a U-PLEX Biomarker Group 1 (mouse) assay (Meso Scale Discovery, Rockville, MD, USA), following the manufacturer's instructions. Samples were incubated in the plate at 4°C overnight to improve detection sensitivity.