Transfection Mode-K cells (luciferase assay) or HEK293 cells (IP-10 overexpression assay) (50% confluent, 24-well plate) were transfected using FuGENE (Roche, Mannheim, Germany) according to the manufacturer’s instructions (24 h). A mixture of 23 ml DMEM (Invitrogen), 1.5 ml FuGENE and 0.5 mg of the appropriate plasmid (pGL3-basic-IP10 (p-IP-10), pGL3-basic (ctr- vector), pIP-10-DsRed, pDsRed-Monomer-C1 (ctr-DsRed)) was added to every well and incubated for 6h. The pGL3-basic-IP10 plasmid was created by cloning a murine IP-10 promoter region (nucleotides 2269 to 228 relative to the start point of transcription) into the XhoI and BglII sites of the pGL3-basic vector (Promega, Madison, WI), placing the promoter directly upstream of the firefly luciferase coding sequence. Therefore, the IP-10 promoter region was amplified from Mode- K cell genomic DNA via pfu (QIAGEN, Hilden) PCR. The sense primer was 59-GCGCTCGAGCTCAAACAGCTCAC-39 (the italic portion indicates the XhoI restriciton site) and the reverse primer was 59-GCGAGATCTTCGAGTGCCGGCTG-39 (the italic portion indicates the BglII restriction site). After digestion and ligation of PCR product and vector, the recombinant plasmid was verified by DNA sequencing (QIAGEN Sequencing Service, Hilden). The PGL3-basic-vector was used as a transfection control. The vector pIP-10-DsRed contains a cytomegalovirus (CMV) promoter sequence driving the expression of the chemokine IP-10, which is N-terminally fused to Red Fluorescent Protein (DsRed). A 0.3 kb cDNA of murine IP-10, covering the whole open reading frame, was amplified from the Mode-K cell cDNA pool by pfu (QIAGEN, Hilden) PCR using primers designed according to the IP-10 cDNA sequence (GenBank Accession No. NM_021274.1). The sense primer for IP-10-DsRed was 59-GCGCTCGAGATAT- GAACCCAAGTGCTGCCG-39 (the italic portion indicates the XhoI restriction site and bold letters indicate the start codon). The antisense primer was 59-GCGCCCGGGAATTAAGGAGCCCTT- TTAGACCT-39 (the italic portion indicates the XmaI site; stop codon in bold letters). The PCR product was digested and cloned into the XhoI and XmaI sites of pDsRed-Monomer-C1 (Clontech, Mountain View, CA). The recombinant plasmid was verified by DNA sequencing (QIAGEN Sequencing Service, Hilden). pDsRed-Monomer-C1 (constantly expressing dsRed) was used as a transfection control. Luciferase assay Transfected Mode-K cells were stimulated with TNF or TNF and L. casei for 24 h. Cells were lysed in 30 ml lysis buffer (Promega) and cell debris were separated by high speed centrifugation. Supernatants (25 ml) were mixed with Reagent A (PJK, Kleinblittersdorf) and the firefly luminescence was measured at 550 nm. A volume of 100 ml of Reagent B (PJK, Kleinblitters- dorf) was added and renilla luminescence was measured at 480 nm. Relative luciferase activity was calculated in percentages: ((firefly/renilla)sample/(firefly/renilla)/control)6100. RNA isolation, reverse transcription and real-time PCR RNA from Mode-K cells or isolated primary IECs was extracted using Trizol Reagent (Invitrogen, Karlsruhe, Germany) according to the manufacturer’s instructions. Extracted RNA was solved in 20 ml water containing 0.1% diethyl-pyrocarbonate. RNA concentration and purity (A260/A280 ratio) was determined by spectrophotometric analysis (ND-1000 spectrophotometer, NanoDrop Technologies, Willigton, USA). Reverse transcription was performed using 1 mg total RNA. Real-time PCR was performed using 1 ml cDNA in a Light CyclerTM system (Roche Diagnostics, Mannheim, Germany) as previously described [27]. Secretional Blockade by VSL#3 Primer sequences were: TLR2; sense 59-TGGGGGTAA- CATCGCT and reverse 59-CATCTACGGGCAGTGG, IP-10; sense 59-TCCCTCTCGCAAGGAC and reverse 59-TTGGC- TAAACGCTTTCAT; 18S; sense 59-CGGCTACCACAT- CCAAGGAA and reverse 59-GCTGGAATTACCGCGGCT. The amplified product was detected by the presence of a SYBR green fluorescent signal. Melting curve analysis was used to document amplicon specificity and crossing points (Cp) were determined. Relative induction of gene mRNA expression was calculated according to the 22DDCp [28] method and normalized to the expression of 18S. Data were expressed as fold change against untreated cells or wildtype mice. Presence or absence of TLR2 transcripts in the transfected HEK293 cells was proven by running a 2% Agarose gel with amplicons (321 bp) generated by reverse transcription of mRNA isolated of the HEK293 cells followed by real-time PCR. T cell transmigration assay Murine splenocytes were isolated from a fresh spleen (mouse genotype: C57Bl/6/N) and activated with 2,5 mg/ml Concavalin A (Sigma) in RPMI (+10%FCS) at 37uC and 5% CO2 for 16 hours. Cells were then centrifuged and about 16108 cells were resuspended in 1 ml RPMI/25 mM Hepes. The cell suspension was then given onto 5 ml NycoPrep 1,077 A (AXIS-SHIELD, Oslo via Progen) and centrifuged (600 g, 20 min). The activated T lymphoblasts which accumulate at the interphase between media and NycoPrep were taken and resuspended in 30 ml washing buffer (RPMI/5%FCS/25mM Hepes). The cells were washed three times (250 g, 10 min) and resuspended to a final cell number of 26106 cells/ml transmigration media (RPMI/5%FCS/25mM Hepes). The activated murine T lymphoblasts were seeded on top of transwell filters (5 mm pore size) at a concentration of 26105 cells/ml transmigration media. The lower chamber was filled with 500 ml of transmigration medium and 100 ml of pure cell culture media or cell culture media from stimulation experiments with Mode-K cells (control, TNF, TNF/fL.c) or TNF-conditioned media supplemented with either a neutralizing anti-IP-10 antibody (30 mg/ml) (R&D Systems) or a control goat-IgG (30 mg/ml) (Dianova). The assay was then incubated for 2 hours (37uC/5% CO2) and the number of transmigrated cells in the lower chamber was analyzed using a CellCounter (Axiovision). The assay was performed in triplicates and the number of transmigrated cells at three representative regions (roi) of each single triplicate was measured. Co-immunoprecipitation Mode-K cells (6-well plates) were stimulated with TNF alone or TNF and L. casei for 6 h. Cells were lysed in 200 ml of 16 lysis buffer (Cell Signaling, Beverly, MA) supplemented with PMSF (1 mM). Cell debris were removed by centrifugation (1400 g, 10 min) and supernatants were incubated with anti-IP-10- antibodies (R&D Systems Europe) (3h, 4uC) in a shaker. Samples were then incubated over night at 4uC in a shaker together with 13 ml of protein A/G beads (Santa, Cruz, Europe), which were previously washed twice with 1xlysis buffer (Cell Signaling, Beverly, MA). Beads were collected by centrifugation (5 min, 8000 g), washed twice with 1xlysis buffer and resuspended in 50 ml Laemmli buffer for subsequent Western blot analysis. Pulse-chase experiment Mode-K cells (6-well plates) in DMEM (0,5% FCS) were stimulated with TNF during the pulse period (S35 Methionine/ Cysteine Labeling Mix, 25 mCi/ml (Perkin Elmer)) (3h). After the pulse period, the cells were washed three times with 16PBS and PLoS ONE | www.plosone.org 3 February 2009 | Volume 4 | Issue 2 | e4365