Gly pro p nitroanilide substrate

After collection of cells for analysis by flow cytometry, peritoneal lavage fluids of NMRI mice were centrifuged at 300 g for 10 min at 4 °C and supernatant was collected and stored at -20 °C. In a flat bottom 96-well plate, lavage fluids (1/2 diluted; 100 µl) were incubated with 500 µM Gly-Pro-p-nitroanilide substrate (Sigma-Aldrich) in 200 mM Tris–HCl buffer (pH 8.3) to determine the soluble CD26 enzymatic activity. Given that sitagliptin is a competitive inhibitor of CD26 and the peritoneal lavage fluids were ½ diluted, the inhibition of the soluble CD26 activity in the peritoneal fluid is underestimated in the CD26 activity assay. Therefore, a calibration curve to estimate the residual CD26 activity was generated, as previously described [107 (link)] (Fig. S7B).

DPP-IV enzyme, Gly-Pro-p-nitroanilide substrate and Coumarin (5,6-benzo-2-pyrone) (C4261) were purchased from Sigma-Aldrich, USA and Quercetin 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-1-benzopyran-4-one dehydrate) RM6194 was obtained from HiMedia Chemicals Pvt. Ltd., Mumbai, India. All the chemicals used in this experiment were of analytical grade, and procured from Merck India Pvt. Ltd. Mumbai, India.

10 μl serum blood samples were diluted in 40 μl phosphate-buffered saline (PBS) pH 7.4 and were mixed with 50 μl of 2 mM Gly-Pro p-nitroanilide substrate (Sigma-Aldrich, St. Louis, MO, USA) to reach a 1 mM final concentration. Once the samples were mixed completely with the substrate, the DPP4 activity was measured spectrophotometrically within 60 min for every 10 min interval at λ = 405 nm at 25°C. Finally, DPP4 activity was calculated using the Beer–Lambert formula [23 ], A = εCl, where A = absorbance, ε = μ molar extinction coefficient (9.45 liters·μmol–1 cm⁻¹ for pNA at 405 nm), C = concentration (μmol·litre-1) and l = length of the light path.