Trypsin Activity Assay

Pancreatic acinar cells were lysed and incubated with butoxycarbonyl-Gln-Ala- Arg-7-amido-4-methylcou-marin hydrochloride (Sigma) in trypsin reaction buffer (10 mM Tris, 20 mM CaCl2, pH 7.4) at 37°C for 30 min. The fluorescence intensity of the reaction solution, which reflects trypsin activity, was measured (excitation: 380 nm, emission: 450 nm) in a fluorescence microplate reader. Purified trypsin protein was added to the reaction mixture separately as the standard. In some experiments, trypsinogen activation was detected using the trypsin fluorescent substrate Rhodamine 110, bis-(CBZ-L-isoleucyl-L-prolyl-L-arginine amide) (Sigma). Briefly, 50 μM of this molecule was added to cells for 20 min, and cells were imaged with microscopy. The fluorescence intensity reflecting trypsin activity was qualified using ImageJ software.