Intestinal homogenates and cytokine measurements

The colon proximal fragment (approximately 1 cm) was transferred to an Eppendorf tube containing 1× Phosphate Buffered Saline solution and Complete ™ protease inhibitor (SIGMA, USA). Then, the fragments were subjected to homogenization in a homogenizer (DREMEL, EUA). The homogenates obtained were centrifuged at 12000 × g for 30 min, and the respective supernatants were stored at -80°C for quantification of BMP2, cytokines, and total proteins. The levels of BMP2, IFNγ, TNF-α, and IL-10 in the proximal intestine homogenates were measured by an enzyme-linked immunosorbent assay (ELISA). For BMP2, the commercial kit PEPROTECH (Lot # 0614T255) was used. For IFNγ (Lot # P209723) and TNF-α (Lot # P210424), the kit used was from the R&D System. Meanwhile, for IL-10, we used the BD OptEIA™ kit (Lot # 9164829). The methodologies were carried out according to the manufacturers’ instructions. For the colorimetric reaction, 3, 3, 5, 5-tetramethylbenzidine (TMB) (BD Pharmingen, USA) was used as a peroxidase substrate and the reading was made on a 450 nm filter in a microplate reader (Bio-Rad 2550 READER EIA, USA). The concentration of total proteins in the intestinal homogenate was determined using a NanoDrop spectrophotometer (Thermo Fisher Scientific, USA) and was used to normalize the concentrations of BMP2 and cytokines. The final concentration was given in pg/mg of tissue.