Macrophage Activation and Cytokine Modulation

On day five, after cell detachment, macrophages were seeded in culture medium without M-CSF, but supplemented with the appropriate stimuli in the following concentrations: 100 ng/ml lipopolysaccharide (LPS, from Escherichia coli, Merck), 5 µg/ml Resiquimod (R848, Enzo life sciences), 100 ng/ml Pam3CSK4 (Biotechne), 5 µg/ml Polyinosinic:polycytidylic acid (poly I:C, In vivogen), 5 µg/ml CPG-c (Enzo Life sciences), 100 ng/ml interferon-gamma (IFNγ, Peprotech), 40 ng/ml interleukin-4 (IL-4, Peprotech), 20 ng/ml interleukin-13 (IL-13, Peprotech), 50 U/ml interferon beta (IFNβ, Peprotech), 20 ng/ml transforming growth factor beta (TGFβ, In vivoGen), 100 ng/ml tumor necrosis factor alpha (TNFα, Peprotech), 100 pg/ml interleukin-10 (IL-10, Peprotech). Due to the volume-per-cell difference between bulk and droplet cultures, the concentrations of stimuli in droplets were adjusted to keep the absolute amount of stimuli-per-cell constant. IL-10 receptor blocking antibodies (αIL-10R, R&D systems), were added to cultures 30 minutes prior to any other stimuli at 6 µg/ml. Cytokine neutralizing antibodies were added during stimulation at the following concentrations: 400 ng/ml IFNβ neutralizing antibody (Biotechne), 100 µg/ml TNFα neutralizing antibody (Adalimumab). Trichostatin A (Sigma Aldrich) was added 30 minutes prior to other stimuli at 1 µg/ml.