Cytation 3 multimode plate imager

DPP4 enzyme activity in mouse serum was measured using H-Gly-Pro-pNA (Sigma-Aldrich, MO, USA) as previously described [18 (link)] Human recombinant DPP4 (Abcam, Cambridge, England) was used as a positive control for DPP4 enzyme activity, and recombinant DPP4 treated with 1µM sitagliptin (Januvia^®) was used as a control for enzyme inhibition. A standard curve was constructed using pNA (Sigma-Aldrich, MO, USA) ranging from 1.56 mM–50 mM. All measurements were carried out in duplicate. Absorbance at 405 nm and 570 nm was measured every 5 min for 3 h at 37 °C using a Cytation 3 MultiMode Plate Imager (BioTek^® Instruments Inc., Winooski, VT, USA). Absorbance values at 405nm were subtracted from readings at 570 nm to account for potential optical interference. Standard curves were constructed using Gen5.0 software v2.05 (BioTek Instruments Inc., Winooski, VT, USA).

The presence of iRFP720-specific IgG antibodies in sera was assessed by ELISA as described [55 (link)]. iRFP720 protein was purified from E. coli cells using a Ni-NTA Fast Start kit (Qiagen, Hilden, Germany). Standards were mouse IgG (Sigma-Aldrich, MO, USA) from 0.5 µg/mL to 0.00781 µg/mL. All measurements were carried out in triplicate. The detection antibody was an Alexa-594-conjugated anti-mouse IgG (5 µg/mL, Abcam). Fluorescence was measured using a Cytation 3 MultiMode Plate Imager (BioTek), with fluorescence (590 nm excitation, 617 nm emission) acquired using a fixed probe height of 6.75 mm, 200 data-points per well and dynamic range 100–80,000 units. Standard curves were constructed using Gen5.0 software v 2.05 (BioTek). A positive IgG serum titre was defined as a reading of >2 standard deviations above the mean for mice implanted with wild-type ID8 cells.