Spleen cell proliferation

On day 5 after colitis induction, mice of all groups were sacrificed and spleens were aseptically removed. The spleens were macerated individually and erythrocytes in cell suspensions were lysed. Cells were pelleted at 200 g for 10 min and cell concentration was adjusted to 1 x 10⁶ cells/mL in RPMI medium (Sigma, USA) supplemented with 10% fetal bovine serum (Cultilab). Cells were stained with 1.25μM Carboxyfluorescein diacetate succinimidyl ester (CFSE) according to manufacturer's instructions (Invitrogen, USA). To determine the maximum uptake, aliquots of the cell suspensions stained with CFSE were fixed with 1% formaldehyde and analyzed by flow cytometer. Stained cells were seeded at 4x10⁵ cell/well in sextuplicate, and Concanavalin A (ConA) was added to each well at final concentration of 2.5μg/mL. Plates were incubated at 37°C in humidified incubator, with 5% CO₂ for 72 hours. After the incubation period, cells were fixed with 1% formaldehyde and proliferation was assessed in CD4⁺CSFE⁺ cells by flow cytometer [34]. Acquisitions were performed with FACScalibur flow cytometer (Becton-Dickinson) and analyzes were done with the FCS Express 5 Plus Research Edition software (FCS Express Launcher). Results were expressed as proliferation index (fold change) calculated in relation to control group. Cells not stained with CFSE were also cultured in the presence of ConA and their supernatants were collected for dosage of cytokines.