Legendplex

Soluble cytokines were measured from blood plasma using a bead-based immunoassay kit as indicated by the manufacturer (Biolegend®; legendplex; 740,389). Samples were run on an LSRII flow cytometer. Data was acquired using FACS DIVA software (BD Biosciences) and analyzed using Biolegend® legendplex software.

Peripheral blood was collected by cardiac puncture at the time the mice were sacrificed and was centrifuged at 13,000 g for 15 min at room temperature. The sera were collected and stored at −80°C until subsequent analyses. Serum cytokines and chemokines were assayed using the multi-analyte flow platform LEGENDplex™ (Biolegend, MN, USA) as per the manufacturer's instruction. The BioLegend LEGENDplex™ panel used in this experiment detects IFN-γ, IL-5, TNF, IL-2, IL-6, IL-4, IL-10, IL-9, IL-17A/F, IL-21, IL-22, and IL-13.

LEGENDplex™ Multianalyte immunoassay for IL-1b, IL-6, IL-8, IFNγ, and SCF was performed in accordance with manufacturer's instructions (LEGENDplex™, BioLegend). In brief, human plasma samples were centrifuged to remove debris and diluted 1:5 to 1:100 in PBS-TB and added to wells containing beads conjugated with analyte-specific antibodies. Detection antibodies were subsequently added to each well. After incubation of the plate for 2 h at room temperature with shaking, streptavidin-phycoerythrin was added, and plates were shaken for an additional 30 min. Finally, beads were washed twice with PBS-T using centrifugation at 1,000 g for 5 min to collect beads after each washing step. Standard solutions containing eight different concentrations of analytes (from 0 to 50,000 pg/mL) were used on each plate for standard curve determination and were incubated the same way as assay samples. After incubation and washing, beads were analyzed using BD FACSAria SORP flow cytometer (BD Biosciences). PMT voltages for Allophycocyanin (APC) and Phycoerythrin (PE) channels were set up immediately before the analysis using the Setup Beads provided in the kit according to manufacturer's instructions. Data analysis and calculations of concentration for samples based on the obtained standard curves was performed using LEGENDplex™ data analysis software according to manufacturer's instructions.

Cytokine levels including free active transforming growth factor (TGF)-β1, granulocyte macrophage-colony-stimulating factor (GM-CSF), interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-2, IL-4, IL-5, IL-6, IL-10, IL-13, and IL-33, in mouse bronchoalveolar lavage fluid (BALF) and serum were assessed using the LEGENDplex (BioLegend) custom panel assay kit as per the manufacturer’s instructions. Cytokine levels were determined using a FACSverse flow cytometer (BD Biosciences), and analyses were performed using the LEGENDplex data analysis software (BioLegend). Total TGF-β1 protein in the culture supernatant or in MSCs was measured using a Human TGF-β1 Quantikine ELISA kit (R&D Systems, Minneapolis, MN, USA) following the manufacturer’s instructions.

T cells (100,000 cells) were cultured alone and in the presence of autologous osteoclasts (20,000 cells) at a ratio 5:1 in 96 well plates. Cells were stimulated with CD3/CD28 mAbs coated beads (Miltenyi Biotech, CA) and media as control. Cultures were performed in the presence or absence of IL-27 (50 ng/ml, PeproTech, NJ). Cells were cultured overnight at 37°C and 5% CO₂ and the supernatants were collected to the analysis of cytokines IFNγ, TNFα, IL-10, IL-17A, and RANKL (LegendPlex™, Biolegend, CA).
For detection of IL-10 secreted by OCs, OCs cultured alone were in vitro stimulated with R848 (10 μM, In vivogen, CA) overnight and IL-10 measured in the supernatant (LegendPlex™, Biolegend, CA).