Functional responses of monocytes and macrophages to TLR ligands

Functional responses of monocytes and macrophages to TLR4 or TLR7/8 ligands were assessed in cell cultures of PBMCs or OptiPrep-isolated IHLs stimulated with either LPS (100 ng/mL, Invivogen) or CL097 (1 μg/mL, Invivogen) in a medium containing BFA (5 μg/mL, Sigma Aldrich), as previously described (29 (link)). Unstimulated cells were used as baseline control. At 16 h post-stimulation, cells were prepared for intracellular cytokine staining following the manufacturer's instructions (FIX & PERM® Cell Fixation & Cell Permeabilization Kit, Thermo Fisher Scientific) and antibodies from Supplementary Table 5. Cells were washed and measured using a BD LSR Fortessa. Further analyses and gating strategies were performed with FlowJo v10 software.