Human il 6

Stattic (BioTechne, 2789) and Tofacitinib (Selleckchem, S2789) were dissolved in DMSO. To inhibit IL-6 signaling, cells were pre-incubated with 1 μM Stattic or Tofacitinib for 1 h at 37°C and 5% CO₂. After pre-incubation, 25 ng/mL human IL-6 (Miltenyi), 25 ng/mL human M-CSF (PeproTech), or BLM-CM (final concentration 50%) was added to the cells where indicated. After a 48 h culture period, cells were harvested and analyzed by flow cytometry. Sunitinib (Sutent, Pfizer) was suspended in DMSO to obtain a 1.43 mM stock solution. To inhibit IL6R and CSF1R signaling, cells were pre-incubated with 10 μg/mL tocilizumab (RoActemra, Roche) and/or 100 nM Sunitinib, or 10 μg/mL Ultra-LEAF Purified anti-human M-CSF antibody (Clone A16067H, Biolegend) for 20 min at 37°C and 5% CO_2. Control samples were incubated with 10 μg/mL mouse IgG2b Isotype control or 10 μg/mL Ultra-LEAF Purified Human IgG1 Isotype control (Clone QA16A12, Biolegend) and/or DMSO as vehicle control. After pre-incubation, 1 ng/mL human IL-6 (Miltenyi), 10 ng/mL human M-CSF (PeproTech), or 50% BLM-CM was added to the cells where indicated. After a 48 h culture period, cells were harvested and divided for phenotype analysis by flow cytometry or re-seeded for allogeneic T cell proliferation assays.