Firefly luciferase reagent

On day 1, Jurkat NFAT-Firefly-Luciferase cells were transfected with the previously described TCR and CAR constructs using standard protocols for the Lonza 4D Nucleofector^™ (AAF-1002B). T2 cells were loaded with the peptides indicated in Table 1. Peptide was resuspended in DMSO, and serially diluted 3-fold twenty times. Serially diluted peptide solutions were added to T2 cells resuspended in peptide-loading media (RPMI1640 + 1% BSA + 1X P/S). This yielded peptide-loaded T2 target cells at approximately 1E6/mL, with peptide concentrations ranging from ~10 fM to 100 μM, including a control at 0 μM. Peptide-loaded T2 cells were incubated overnight at 37°C in 384-well plates (Thermo Scientific AB0781). On day 2, the cells were cocultured in a 384 well plate (Corning 3570). Peptide-loaded T2 cells (10,000 cells/well) were added to transfected Jurkat-NFAT-Firefly-Luciferase cells (12,000 cells/well) to a final volume of 20 μL. After a 6-hour incubation at 37°C, 20 μL firefly luciferase reagent (BPS Bioscience 60690–1) was added to each well and incubated for 10 minutes in the dark. Luminescence was monitored on the Tecan Infinite^® M1000 with a 100 ms integration time.

NKA and the peptide analogs of NKA listed in Table 1 were synthesized by Genscript (Piscataway, USA) to a purity ≤95%. NKA and substance P were purchased from Sigma Aldrich, (St. Louis, USA); septide was purchased from Bachem (Bubendorf, Switzerland, EU). [³H]-Septide was custom synthesized from Quotient Bioresearch (Cardiff, UK). [¹²⁵I]-NKA, and Microscint 20, were obtained from Perkin Elmer (Boston, USA). F12K medium, Geneticin (G418) and Lipofectamine 2000 were obtained from LifeTechnologies (Carlsbad, USA). Phosphate buffered saline (PBS) was from Lonza (Walkersville, MD, USA) and EDTA from Life Technologies (New York, USA). FuGENE HD Transfection Reagent was obtained from Promega (Madison, USA). The following reagents were obtained from Sigma Aldrich (St. Louis, USA): DMSO, Pluronic F-127, HEPES, bacitracin, SIGMAFAST Protease Inhibitor Tablets, bovine serum albumin (BSA), polyethileneimine (PEI), NaCl, 3-isobutyl-1-methylxantine (IBMX), and probenecid. The CRE/CREB Reporter Assay Kit, Firefly Luciferase reagent, and Renilla Luciferase reagent were purchased from BPS Bioscience (San Diego, USA).