Full details of the methods are given in Additional File 3 The e

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 chosen based on the findings Selleckchem URMC-099 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 PI3K inhibitor 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 Terminal deoxynucleotidyl transferase 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 LY294002 acknowledge the contribution of Kelly Armstrong (fluorescent microscopy) and Paul Maclean (gene ontology and KEGG pathway analysis).

Comments are closed.