Full details of the methods are given in Additional File 3. The expression of tight junction-related genes differentially expressed from the microarray analysis was confirmed using qRT-PCR. The expression of seven target genes relative to three reference genes was assessed using the standard curve method. The reference genes (GAPD, SDHA and YWHAZ) were TPX-0005 clinical trial chosen based on the findings INK1197 clinical trial of Vandesompele et al [52] and their log ratios in the microarray data (close to 1; not differentially expressed). Five target genes (ZO-1, ZO-2,

OCLN, CGN and ACTB) were chosen from the tight junction-related genes that were differentially expressed (all up-regulated) in the microarray analysis. The two other target genes, GJA7 and CLDN3, were chosen to be included because they were down-regulated and not differentially expressed, respectively,

in the microarray analysis. The analysis was carried out as described in Additional File 3 and the data was analysed using Relative Expression Software Tool 2008 (version 2.0.7) with efficiency correction [53]. Fluorescent microscopy Caco-2 cells were grown on Lab Tek II Chamber Slides with Permanox™ coating (Nalge Nunc International Corp, Naperville, IL, USA) for 6 days until confluent. Caco-2 cells were treated with L. plantarum MB452 (OD 600 nm 0.9) or control media for 8 hours (n = 4 per treatment per antibody). After treatment, Caco-2 cells were rinsed twice with selleck products PBS, fixed in either 4% (w/v) paraformaldehyde for 20 minutes (for CGN and ZO-1) or ice cold 70% ethanol (for ZO-2 and OCLN), quenched with 50 mM NH4Cl (in PBS) for 15 minutes, and blocked with blocking solution (2%

(v/v) foetal bovine serum, 1% sheep serum albumin, 0.1% Triton X-100, 0.05% Tween 20 in PBS, pH 7.2) for 20 minutes. Caco-2 cells were then immuno-stained with the primary antibodies (2.5 µg/mL rabbit Phloretin anti-ZO-1, 1.25 µg/mL rabbit anti-ZO-2, 2.5 µg/mL rabbit anti-occludin, 1 µg/mL rabbit anti-cingulin; Zymed, Invitrogen, NZ) in blocking solution for 1 hour, followed by a PBS wash (0.1% Triton X-100, 0.05% Tween 20 in PBS) to reduce non-specific staining, and the secondary antibody, Alexa Fluor 488 goat anti-rabbit IgG (5 µg/mL for ZO-2, 10 µg/mL for rest; Invitrogen, NZ) in blocking solution for 1 hour. The slides were imaged with a fluorescent microscope (Leica DM2500 microscope, Leica DFC420C camera) with the following settings: exposure 1.1 ms, saturation 2.25, gamma 1.52, gain 8.4× and magnification 40×. The images were viewed using LAS Image Overlay software (Leica Application Suite v1.8.2). Acknowledgements This work was funded by the AgResearch Internal Investment Fund. RCA is funded by a New Zealand Foundation of Research, Science and Technology Postdoctoral Fellowship (AGRX0602). The authors acknowledge the contribution of Kelly Armstrong (fluorescent microscopy) and Paul Maclean (gene ontology and KEGG pathway analysis).