Cpg oligonucleotide odn1826

We used two strategies to test the role of TLRs in microglial response to K. variicola. After cells were seeded into 24-well plates and incubated in RPMI supplemented with 2% FBS and 1% penicillin–streptomycin for 24 h, first, we targeted TLR2. Microglial cells were pre-treated for 1 h with either TLR2 antagonist (T2.5) at a concentration of 10 µg/mL (InvivoGen) or TLR2 agonist, lipoteichoic acid (LTA-SA) at a concentration of 10 µg/mL (InvivoGen). Then, we targeted TLR9, which is downstream of TLR2. We pre-treated the microglial for 1 h with TLR9 antagonist (ODN2088) at a concentration of 1 µM (InvivoGen) before introducing K. variicola. In parallel, we used the TLR9 agonist, CpG oligonucleotide (ODN1826), at a concentration of 1 µM (InvivoGen). These conditions are shown in Additional file 1: Table S3.

The interaction of CLR agonists with ovalbumin was determined by surface plasmon resonance (SPR) using a Biacore 3000 (GE Healthcare, Uppsala, Sweden) equipped with a research-grade CM5 sensor chip (BR100012, GE Healthcare) at a temperature of 25°C. Ovalbumin was immobilized at amount of 30,000 response units (RUs) in 10 mM acetate buffer pH = 5.0 on a CM5 sensor chip using the amine coupling kit supplied by the manufacturer (GE Healthcare, Sweden). TDB, curdlan, and furfurman were used at concentration of 100 μg/mL, 50 μg/mL, and 25 μg/mL. As a control of nonspecific binding of agonist with antigen, we used CpG oligonucleotide ODN1826 (InvivoGen, USA) at a concentration of 100 μg/mL. PRR agonists were dissolved in sterile deionized water containing 10% v/v DMSO. Analyses were performed in 10 mM HBS-EP running buffer (HEPES buffered saline containing 150 mM NaCl, 3 mM EDTA, and 0.005% surfactant P20, pH 7.4) at a flow rate of 30 μL/min. Calculations were performed using BIAevaluation software (GE Healthcare, Uppsala, Sweden) with reference-subtracted fitting. Representative images of SPR are shown in Figure
S2.