Cytokine Profiling of Monocyte Subsets

CD11b⁺-enriched cells (10,000–20,000 cells/well) from 27 samples were cultured in RPMI-1640 media supplemented with 10% fetal bovine serum, 10,000 units/mL of pen/strep, 1% GlutaMAX (Thermo Fisher Scientific, Waltham, MA), and 5 ng/mL GM-CSF. Six different experimental conditions were tested per sample: (1) unstimulated cells; (2) stimulated with 10 μg/mL LPS; (3) 1 μM^{11 (link),54 (link)} ruxolitinib (InvivoGen, San Diego, CA); (4) 1 μM tofacitinib (Invivogen); (5) 10 μg/mL LPS 1 mM ruxolitinib; and (6) 10 μg/mL LPS + 1 μM tofacitinib. After overnight incubation, supernatants were collected for cytokine quantification by Luminex using a Human High Sensitivity Cytokine customized premixed magnetic bead panel (R&D Systems, Minneapolis, MN) for the following: IL-1 β/IL-1F2, IL-6, IL-8/CXCL8, and TNF. The median fluorescence intensity data were analyzed using MILLIPLEX Analyst Software V.3.5 (MilliporeSigma), and concentrations were calculated in pg/mL in relation to an R&D standard.