Butyrate-induced differentiation in CaCo-2 cells

Human colorectal adenocarcinoma CaCo-2 cells were grown in Dulbecco’s modified Eagle's medium (Gibco, Thermo Fisher Scientific) with 10% FCS. Cells were subcultured at 80% confluency and maintained at 37 °C in a humidified incubator with 5% CO₂. At day 0, when cells reached full confluency, butyrate was added to the appropriate cells (at 2 mM final concentration in the cell culture flask). For the treated cells, butyrate was added to the medium at each medium exchange. Cells were grown in three separate culture flasks (three biological replicates) for both butyrate-treated and spontaneously differentiated group. On days 5, 7, 14, 21, and 24, cells were collected. Prior to harvesting the cells, medium was removed and adherent cells were washed twice with DPBS and trypsinized using 0.25% trypsin–1 mM EDTA. To stop the trypsin activity, medium (without FCS) in a ratio of 2:5 (trypsin:medium; v/v) was added, and cells were pelleted at 300g for 5 min. Cells were resuspended in DPBS and counted and aliquoted to ∼2.0 × 10⁶ cells per replicate and washed twice with 1 ml DPBS for 3 min at 100g. The supernatant was removed, and cell pellets were stored at −20 °C until further use.