Birb796

Gonads were dissected in Hepes-Dulbecco Modified Eagle Medium (DMEM; Invitrogen, Carlsbad, CA) supplemented with 15% fetal bovine serum (FBS; Hyclone Laboratories, Logan, UT). PGCs were cultured in high-glucose DMEM supplemented with 0.1 mM nonessential amino acids, 0.1 mM 2-merkaptoethanol, 100 IU/ml penicillin, 100 μg/ml streptomycin, 2 mM glutamine, 1 mM sodium pyruvate, and 15% FBS. Isolated PGCs were treated with 0.2–100 ng/ml FGF9 (Sigma-Aldrich, St. Louis, MO). In some experiments, PGCs were cultured with p38 inhibitors (BIRB796; Cayman Chemical Company, Ann Arbor, MI, SB202190; Sigma-Aldrich) at 10 μM or MEK inhibitor (U0126; Cayman Chemical Company) at 1 or 10 μM.

Assays were carried out with approximately 1 to 2 × 10⁴ cells/well of microglia seeded in 24-well tissue culture plates. At the time of the assay, the culture medium was removed and replaced with fresh medium without antibiotics, and the cells were pretreated with the pertinent inhibitors for 2 h prior to adding B. burgdorferi (MOI of 10:1). The microglial cells were incubated with the bacteria and inhibitors for a further 24 h, followed by collection of supernatant after centrifuging at 2,095 × g for 10 min at 4°C. Supernatants were stored at −20°C until analysis. The following inhibitors were used: SB203580 and BIRB796 (p38); U0126 (MEK1/2); SP600125 and Jun N-terminal kinase (JNK) inhibitor VIII (JNK) (all but one were from EMD Millipore, Billerica, MA; BIRB796 was obtained from Cayman Chemical Co., Ann Arbor, MI); and oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC) (TLR2/4), CLI-095 (TLR4), Gefitinib (RipK2), and myeloid differentiation primary response 88 (MyD88) inhibitory peptide (InvivoGen, San Diego, CA).
Pam3CSK4 (Imgenex, San Diego, CA), LPS O55:B5 (Sigma-Aldrich, St. Louis, MO), or muramyl dipeptide (MDP, InvivoGen) were included as positive control agonists when required.