Tc treated microplates

One day prior to assay, 5,000 PSCs/well were added to 96 well flat clear bottom white polystyrene TC-treated microplates (Corning, cat#3610). The following day, PSC media was aspirated off and 50 μL of NK92 cells (lacking GFP) were added to each well containing PSCs at a 4:1 E:T ratio and incubated overnight at 37°C. 100 mM stock of dipeptidylpeptidase substrate (Acetyl-Aka-Gly-Pro-AFC) (Anaspec, CatAS-24126) was made by resuspending lyophilized substrate in DMSO. On the day of the assay, DMSO stock was then diluted 1:1000 in FAP activity assay buffer (50 mM Tris-BCl, 1 M NaCl, 1 mg/mL BSA, pH 7.5). A standard curve was generated using rFAP (R&D systems, 3715-SE-010). 50 μL of rFAP standard ranging in concentration from 0.03125–2ug/mL was added to wells in triplicate. 50 μL of substrate was added to each well and the plate was incubated for 5 minutes at 37°C. The plate was read on a PerkinElmer EnVision Multimode Plate Reader with 390–400 nm excitation and 580 – 510 nm emission wavelengths. The final concentration of FAP per well was calculated using the standard curve. Data were compiled and assessed for statistical significance using GraphPad Prism 9.

Ninety-six-well TC-treated microplates (Corning, USA) were planted with 4000 cells/well. Cells were counted by TC20 Automated Cell Counter (Bio-Rad, Hercules, CA, USA). Cells were incubated for 72 h, after which 1/10 of the medium volume of MTS reagent (Promega, Madison, WI, USA) was added to each well. After 90 min of incubation at 37 °С and 5% СО₂, the absorbance was measured by SPECTROstar Nano (BMG LABTECH, Ortenberg, Germany) at 495 nm.