Microbca kit

After sorting, SP cells and MP cells were washed several times with PBS and frozen at −80 °C. For lectin microarray analysis, 1 × 10⁵ cells were resuspended in 1 ml of PBS-T (containing 1% TritonX-100), followed by sonication for 15 min and centrifugation at 14,000g for 15 min. The supernatant was recovered. Proteins were quantitated using a micro BCA kit (Thermo Scientific) and labelled with fluorescent dye Cy3 (Thermo Scientific). Samples with protein concentration of 250 ng/ml were applied to a LecChip (Glyco Technica) and incubated at 20 °C for 16 h. The chip was then scanned with a GlycoStation Reader 1200 (Glyco Technica) confocal scanner. Each lectin in LecChip has three replicates. To be normalized, intensity of each well in lectin microarray was divided by the mean of total 135 wells’ intensity of the chip. We repeated lectin microarray analysis of SP and MP cells using independent samples to overcome biological bias.

Tamoxifen, 4-hydroxy-Tamoxifen, Dimethylsulfoxide, 2’,7’-dichlorofluorescin diacetate, 5,5’-dithio-bis (2-nitrobenzoic acid), colchicine, valspodar (PSC-833) and Tween-20 (P9416) were all purchased from Sigma Aldrich. Lipofectamine 2000, the microBCA kit and the Pierce™ ECL Western Blotting Substrate Kit were all products of Thermo Fisher. Propidium iodide and the HRP-conjugated goat anti-mouse were purchased from Invitrogen, and the IncuCyte® is a product of Essen BioScience.

To examine the effect of phage EK99P-1 on tight junction proteins, 1 × 10⁵ cells were cultured on 12-well plates for 2 days until confluence was reached. Confluent IPEC-J2 was treated with ETEC K99 and/or phage EK99P-1, washed with PBS and then lysed in a RIPA lysis buffer (50 mM Tris–HCl, 150 mM NaCl, 1% NP_40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate containing protease inhibitor), followed by protein quantitation using a Micro BCA kit (Thermo, USA). The same amount of extract was loaded into 12% Tris–glycine polyacrylamide gel and electrophoresed. Then, the proteins were transferred onto a polyvinylidene fluoride microporous membrane for 90 min at 4 °C and blocked with 5% skim milk in TBS-T (1 M Tris–HCl, 5 M NaCl, 10% Tween-20) for 1 h. The blot was incubated with rabbit anti-claudin-3, -occludin, and -ZO-1 or mouse anti-β-actin antibodies (Invitrogen, USA) overnight. Then, the membrane was washed and incubated with goat anti-rabbit or anti-mouse IgG-HRP antibody (Santa Cruz Biotechnology, USA) for 1 h. The target protein was visualized using an enhanced chemiluminescence system (GE Healthcare, USA), followed by analysis using a ChemiDoc XRS system (Bio-Rad, USA).

A preparation containing 1,300 μg/mL of whole viral purified particles of O1/Campos, preserved in aliquots at -80°C, was either used directly to coat plates with PBS or carbonate/Bicarbonate buffer (pH: 9.6) or treated at 60°C for 20 minutes to produce 12S particles, as described before [17 (link)]. Different ratio of 146S to 12S particles were prepared and used to coat duplicate wells. The absence of 146S particles in the heated preparations was confirmed by sucrose-gradient ultracentrifugation. Purified IgG at the same concentration was used as control. ELISA plates were incubated overnight at 4°C, following the standard procedure. The next day, plates were washed five times with PBS-Tween 20 (0.05%) and bound total proteins were quantified using Micro BCA kit (Thermo Fisher) following the manufacturer´s instructions.
The same design was used to run 10 serum samples from 60 dpv vaccinated animals (diluted 1: 50) comparing the OD values for each captured antigen preparation with that of the whole particles (expressed as percentage of residual reactivity)