Active DPP IV in Biological Samples. Cells (cell types are denoted in the figure) were cultured in DMEM with 10% FCS and harvested at 80-90% confluency. Total cell lysates were prepared using CytoBuster™ Protein Extraction Reagent (Cat. No. 71009) according to the manufacturer's recommended protocol. Human spleen extract was prepared by homogenizing tissue with 170 mM NaCl, 2 mM EDTA. Human plasma (pooled samples) was obtained from commercial sources. The concentration of active DPP IV was determined by extrapolating the RFU values to the standard curve prepared with human recombinant DPP IV. Protein concentration in cell lysates and tissue extract was determined using a BCA protein assay. Activity was assessed as outlined in the Detailed Protocol above. Fluorescence intensity was measured at an excitation wavelength of 360 nm and an emission wavelength of 460 nm and expressed per mg total protein.

Activity of Human Recombinant DPP IV. Human recombinant DPP IV activity was measured as outlined in the Detailed Protocol above using a 1-h incubation at 37°C. The data is presented as Relative Fluorescence Units.

Cross-Reactivity. The activities of recombinant human DPP II, FAP, and DPP I were assessed as outlined in the Detailed Protocol above.

Storage Conditions.