2.3. Determination of DPP-4 Activity in Caco-2 Cells

Caco-2 cells were seeded into 96-well plates at a low density (1 × 10⁵ cells/mL) and high density (7 × 10⁵ cells/mL), respectively. The DPP-4 activity in the cells was measured on the 2nd, 4th, 7th, 10th, 14th, 18th, and 21st days after seeding. For determination of the DPP-4 activity, after washing the cells in each well with PBS, 100 µL of Gly-Pro-PNA·HCl at a concentration of 4 mM was added and the plate was incubated at 37 °C for 60 min. Afterwards, the absorbance (Abs) of the reacted samples was measured at 405 nm in a SYNERGY H1 microplate reader (BioTek, Winooski, VT, USA).

In order to determine the culture time of Caco-2 cells, the inhibitory effect of sitagliptin on DPP-4 was determined. The effect of sitagliptin on the DPP-4 activity was assayed using a previously reported method [19]. The Caco-2 cells were seeded into 96-well plates at a density of 7 × 10⁵ cells/mL, and the DPP-4 activity was measured on the 2nd, 4th, 7th, and 10th days after seeding. Before determination of the DPP-4 activity, the cells in each well were washed with PBS, and 100 µL sitagliptin in PBS at concentrations of 10⁻⁴, 10^−3.5, 10⁻³, 10⁻², and 10⁻¹ mM were added separately, and the plate was incubated at 37 °C for 30 min. The PBS solution was regarded as the positive control. The sample control and negative control were the same as the sample group and positive group, respectively, except that no cells were seeded. Then, the cells in each well were washed with PBS to completely remove the treatment medium. Subsequently, 100 µL Gly-Pro-PNA∙HCl at a concentration of 4 mM was added to each well and was incubated at 37 °C for 60 min. Afterwards, the Abs of the reacted samples was measured at 405 nm in a SYNERGY H1 microplate reader (BioTek). The inhibition rate of DPP-4 (DIR) was calculated using the following Formula (1).