Spectramax m3 system

Sulphated polysaccharide concentration was initially estimated in samples and extracts through interaction with DMMB dye. 200 μL DMMB dye solution was added to 25 μL samples, blanks or standards (chondroitin sulphate from bovine trachea, Sigma-Aldrich, Castle Hill, NSW, Australia) in triplicate, in a 96 well plate (Nunclon Delta Surface, Thermo Fisher Scientific, Waltham, MA, USA). The plate was mixed for 1 min using a plate mixer (IKA^® MS digital 96 Well Plate Mixer, Staufen im Breisgau, Germany) and absorbance was measured at 525 nm using a Spectra-Max M3 System spectrophotometer (Molecular Devices, Sunnyvale, CA, USA). Sulphated polysaccharides were calculated from the standard curve using SoftMax-Pro 6.1 software (Molecular Devices, Sunnyvale, CA, USA).

IgA concentration was measured using Bovine IgA ELISA Quantitation Set (Bethyl Laboratories, Montgomery, USA) following the manufacturer’s instructions. The intestinal content samples were centrifuged and the supernatants were collected for further measurements. A reference standard of bovine serum, containing 0.11 mg/ml IgA was used to calibrate the assay for the samples. The absorbance was measured with the SpectraMax M3 system (Molecular Devices, Sunnyvale, USA).

Glycogen concentration was measured using Glycogen Assay Kit (Cayman Chemical, Ann Arbor, MI), according to the manufacturer’s instructions. Briefly, the frozen tissues were minced into small pieces, and then homogenized to disrupt cells. Homogenate was centrifuged and 50 μl of supernatant was used for glycogen measurement. The reagents of Glycogen Assay Kit (Cayman Chemical) were able to hydrolyze glycogen and generate highly fluorescent product resorufin (Glycogen Assay Kit Manual). Fluorescence (Ex/Em = 535/587 nm) was measured with the SpectraMax M3 system (Molecular Devices, Sunnyvale, CA).