Culture supernatants were clarified by filtering through 0.22-μm filter (SLGV004SL; Millipore) and stored at −80°C until use. BM fluids were prepared as previously described (Kang et al., 2020 (link)) and stored at −80°C until use. For ELISA measurements, 50 µl of culture supernatants were analyzed with IL-6 (50-172-18; eBioscience) or TNFα (88-7324-22; eBioscience) ELISA kits according to the manufacturer’s instructions. For Luminex cytokine multiplex bead arrays, 25 µl of culture supernatants were analyzed for a custom-made panel of five cytokines (IL-1β, IL-6, GM-CSF, TNFα, MIP1α; Invitrogen, PPX-05) according to the manufacturer’s instructions. For Raybiotech 200 mouse cytokine arrays, 500 µl of pooled culture supernatants or BM fluids were sent per sample to Raybiotech for quantitative proteomics services using Mouse Cytokine Array Q4000 kit. Quantile normalization was performed for direct comparison of secreted cytokine profiles from different array experiments using R Bioconductor package, and hierarchical clustering was conducted using pheatmap package of R (https://cran.r-project.org/web/packages/pheatmap/index.html ). The statistical significance of the different distributions of secreted cytokine under diverse biological conditions was determined using Kruskal-Willis test (P value <0.05, R version 3.3: https://www.r-project.org ).
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