Fast prep 24 5g machine

Frozen ileum tissue (ca. 100 mg) was homogenized with cell lysis buffer (500 μl) (Cat EPX-99999-000 eBioscience, San Diego, USA) with 0.1 % proteinase inhibitor cocktail (Sigma, St. Louis, MO) and 1 % NP40 in ceramic bead tubes using Fast Prep-24™ 5G machine (M.P. Biomedical LLC, California, USA) at speed 6.0 msec for 30 s (twice), and then centrifuged at 12,000 g for 15 min at 4 °C. Supernatants were collected and 100 μg of protein was used for Western blot analyses. Membranes were probed with 1:1000 dilution of rabbit anti-VE-cadherin, rabbit anti-occludin, and rabbit anti-claudin 3 (Abcam, Cambridge, MA) primary antibodies and subsequently incubated with 1:10,000 dilution of goat-anti-rabbit HRP (BioRad Laboratories Inc., California). Detection was performed using a chemilumiescence system (super signal west chemiluminescent substrate, Thermo Scientific). Immuno-quantitation was performed by densitometric scanning of the blot and normalized against the signal from β-actin (Sigma Aldrich, St Louis, MO) using Image Quant software (Image Quant TL 8.1 Version). We measured calcium levels in urine from breast-fed and formula fed piglets using colorimetric assay from Bio Vision (Catalog#K380-250) as per manufacturer’s instructions.

Expression of IL-8, IL-6, and IL-10 were measured in ileum samples of pigs using porcine ELISA kit (EMD Millipore Corporation, MA) as per the manufacturer instructions. Frozen ileum tissue (100 mg) was homogenized with cell lysis buffer (500 μl) (Cat EPX-99999-000 eBioscience, San Diego, USA) in ceramic bead tubes using Fast Prep-24™ 5G machine (M.P. Biomedical LLC, California, USA) at speed 6.0 msec for 30 s, and then centrifuged at 12,000 g for 15 min at 4 °C. Clear tissue supernatant were used to measure the protein expression. Tissue cytokines data was normalized with total protein estimated by BCA method (Thermo-scientific IL, USA) and data are presented as pg/g protein.