Regierungspraesidium Karlsruhe (written permit #T4/08). TLR22/2 [13] and MyD882/2 mice [14] were backcrossed to C57Bl/6 background for .6 generations. For B cell isolation spleens were passed through a mesh, filtrated and B cells isolated with anti-B220 (Fig. 1D) or anti-CD19 microbeads (Fig. 1E) (Miltenyi Biotec, Bergisch-Gladbach, Germany). B cell purity was 9563%. B cells were resuspended in culture medium (RPMI 1640 (Invitrogen, Karlsruhe, Germany) supplemented with 10% FCS (BioWest, Nuaille, France), 1% Penicillin-Streptomycin, 1% HEPES buffer and 1:50000 b-mercaptoethanol (all from Sigma, Munich, Germany)). Human PBMC and B cell isolation In accordance with the declaration of Helsinki venous blood was drawn from healthy volunteers after verbal and written information on potential adverse events of the blood draw, research goals and measures securing the anonymity of the donors. We obtained written informed consent before venous puncture. The procedure was approved by the ethics commission of the Medical Faculty of the University of Heidelberg, Germany (written approval #157/2006). PBMC were isolated from heparinised venous blood by Ficoll gradient centrifugation. B cells were isolated with anti-CD19 microbeads (Miltenyi). B cell purity was 9861%. B cells were resuspended in culture medium and incubated over night before stimulation to obtain resting B cells. Cell Stimulation The stimulatory reagents were used at the following concentra- tions, unless otherwise indicated: all ODN were used at (1 mM) and purchased from MWG Biotech, Munich Germany: CpG ODN 1668 PTO (59-tccatgttcctgatgct-39; full-length PTO), CpG ODN 2006 PTO (59-tcgtcgttttgtcgttttgtcgtt-39; full-length PTO), GpC ODN 2006 PTO (59-tgctgcttttgtgcttttgtgctt-39; full-length PTO), CpG ODN 2006 PO (59-TCGTCGTTTTGTCGTTTTGTCG- TT-39); Pam3CSK4 and FSL-1R (HEK293 and monocytes: 200 ng/ml; B cells: 1 mg/ml)) (EMC Microcollections, T u¨bingen, Germany), MALP-2 (HEK293: 25 ng/ml; B cells: 1 mg/ml) (Alexis Biochemicals, Loerrach, Germany), highly purified LPS from Salmonella (gift from U. Seidel, Research Center Borstel, Germany), SpA (Amersham, Uppsala, Sweden), anti-human IgM+IgG+IgA F(ab9)2 fragments (Dianova, Hamburg, Germany), PMA (1 mM) and ionomycin (0.5 mg/ml) and polymyxin B (250 U/ml), all from Sigma. The PWM preparations used in this study were purchased from Sigma (Lot. O L8777-5MG (Lot. # not available, purchased in 2005), Lot. A: L8777-5MG #045K7550, Lot. B: L8777-5MG #077K76801, Lot. C: L9379-40MG #038K7680V) and from EY Laboratories, San Mateo, CA, USA (Lot. D: L1901 #260529-5) and used at 1 and 10 mg/ml as indicated. The tomato (Lycopersicon esculentum)-derived lectin was purchased from EY laboratories (Lot# 270624-1). As for PWM Lot. D this lot was nearly free of contaminants in LAL, 16 s rDNA PCR and TLR2 assays (data not shown). For neutralisation of human TLR2-transfected HEK293 cells and B cells were pretreated with 10 mg/ml anti-TLR2 mAb for 30 minutes (Genentech, South San Francisco, CA, USA) or the respective isotype control, i.e. murine IgG1k (Dako, Hamburg, Germany). For DNAse treatment 20 ml PWM stock 0.1 mg/ml were incubated with or without 1 unit RNAse-free DNAse (Roche, Mannheim, Germany) and 2 ml 106 incubation buffer provided by the manufacturer over night at 37uC. For enzyme inactivation probes were heated to 75uC for 10 minutes and then frozen until PWM-Induced B Cell Activation further use. N,N-di-acetylchitobiose and N,N,N-tri-acetylchito- triose were purchased from Sigma and used at 400 mg/ml. Proliferation assays For analysis of 3H-thymidine incorporation B cells were plated at 0.25610*6/ml and stimulated for 72 hours, and pulsed 16– 18 hours before harvest with 1 mCi/well 3H-methyl-thymidine. Proliferation was quantified by detected counts per minute (cpm). For CFSE dilution assays B cells were stained with 10 mM CFSE (Invitrogen), plated at 1610*6 B cells/ml, stimulated and analyzed by flow cytometry on day 4 or 5. Transfection of HEK293 cells and IL-8 ELISA For IL-8 measurements HEK293 cells were transfected with pTLR2 [15] 200 ng/well complexed with lipofectamine 2000 (Invitrogen) as previously described [7]. Cellular supernatants were harvested 24 hours after stimulation and analyzed for IL-8 by ELISA (BD Opteia, BD Bisociences, Heidelberg, Germany). LAL assay Endotoxin content was determined by kinetic chromogenic limulus amoebocyte lysate (LAL) test (Bio Whittaker, Verviers, Belgium) performed according to the supplier’s instructions. Immunofluorescence For immunofluorescence analysis 0.5610*6/well HEK293 cells were grown over night on poly-lysine D (Sigma) coated coverslips in 24-well plates. Cells were transfected with pTLR2 or with lipofectamine alone and stained for TLR2 24 hours after transfection. For immunofluorescence analysis cells were fixed with PBS/4% PFA, stained with anti-TLR2 mAb or the murine IgG1k isotype control in Fix & Perm Medium B (Invitrogen), mounted in 0.1 mM DAPI (Invitrogen)-containing PBS/glycerol and stored at 4uC until analysis on a Leica DMI 6000B (Leica Microsystems, Mannheim, Germany). Flow cytometry All experiments were performed on a FACSCanto device (BD Biosciences) and analyzed using the FACS Diva software. Only live gated cells were subjected to analysis. Analysis of intracellular TNF expression in human monocytes was performed as previously described [16]; briefly, PBMC (2610*6/ml) were incubated with stimulatory reagents with/without Brefeldin A (1 mg/ml, Sigma) for 4 hours, fixed, permeabilized, stained with PE-conjugated anti- human TNF (Becton Dickinson) and analyzed by flow cytometry. Monocytes were gated based on forward scatter (FSC)/side scatter (SSC) properties. Amplification of bacterial ribosomal DNA and sequencing Isolation of DNA was performed with the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany). For amplification of 16S ribosomal DNA PCR was performed with the following degenerate primers: forward 59-AGA GTT TGA TCM TGG CTC AG-39; reverse 59-CCG TCA ATT CMT TTR AGT TT-39 and the FastStart TaqPolymerase Mix (Roche, Mannheim, Germany). Annealing: 53uC 1 min., Extension: 72uC 1.5 min., 35 cycles. The expected fragment size (E. coli) is 919 bp. 16S rDNA-PCR products were purified using the PCR purification kit from Qiagen, Hilden, Germany and sequenced at GATC Biotech AG, Konstanz, Germany. Sequences were blasted against the NCBI database. PLoS ONE | www.plosone.org 2 January 2012 | Volume 7 | Issue 1 | e29806