Immobilized tpck trypsin

The Caco-2 cells were routinely grown at 37°C in a 95% air–5% CO₂ atmosphere in Dulbecco's modified Eagle medium (DMEM) with high glucose (Sigma-Aldrich Co) and 10% heat-inactivated (56°C, 30 min) fetal bovine serum (FBS; Gibco, Grand Island, NY, USA). The cells were harvested using trypsin-EDTA (Gibco) and suspended with DMEM-10% FBS to stop the reaction with trypsin-EDTA. The cells were collected from the cell suspension by a centrifugation, and were washed with Dulbecco's phosphate buffered saline (DPBS; Gibco). Then, the cells were resuspended with DMEM without any serum, and the mixture was centrifuged twice. Finally, the cellular suspension in DMEM was adjusted to 1.0 × 10⁵ cells per 75 cm² of the tissue culture flask (Thermo Scientific, Waltham, MA, USA). The culture medium was replaced every 5 days, and the supernatant collected after 5, 10, and 15 days of culture was used in subsequent experiments. The collected supernatant was filtered with Centriprep YM-3 or YM-10 (3 or 10 kDa; Millipore, Bedford, MA, USA). For some experiments, 500 μl of the various Caco-2 cell supernatant samples were treated with 25 μl of Immobilized TPCK Trypsin (Thermo Fisher) for 1 h at 37°C; the Immobilized TPCK Trypsin was then removed by centrifugation (10,000 × g for 10 min).