Investigating Prevotella-induced Cytokine Response

Bone marrow-derived dendritic cells (BMDCs) were obtained as previously described [30 (link)]. Briefly, bone marrow cells were isolated from 6- to 8-week-old C57BL/6 mice and cultured for 9 days with 20 ng/mL recombinant murine GM-CSF (PeproTech). Cells were purified by positive selection [26 (link)] using anti-CD11c microbeads (Miltenyi Biotec). The dendritic cell (DC) phenotype was controlled by flow cytometry. The cytokine response to Prevotella was assessed in 1 × 10⁵ BMDCs incubated with LPS (10 ng/mL, Sigma) as a positive control, PBS as a negative control, or heat-killed (30 min at 60°C) Prevotella (1 × 10⁷ cfu/well) for 24 h [15 (link)]. To compare the efficacy of Toll-like receptors (TLRs), the BMDC medium was supplemented with inhibitors of TLR2 [31 (link)] (C29, MCE, 50 μM) or TLR4 [32 (link)] (TAK-242, MCE, 100 nM). For all experiments, TLR inhibitors were added to the cells just prior to stimulation.