Nano glo luciferase

SEV-donor cells SK-N-AS and A549 were seeded at a density of 5 × 10⁵ cells in a 6-well plate the day before transfection. Then, cells were transfected with 2 µg Nano-Luciferase (NLuc)-Hsp-70 plasmid (Bonsergent et al., 2021 (link)) and Lipofectamin 2000^® according to the manufacturer’s instructions 18 h before sEV harvesting. SEV were harvested from the supernatant of A549 or SK-N-AS cells. 2 μg sEV were blocked with 200 ng as previously described in a total volume of 40 µL for 21 h. Acceptor cells were seeded in 96-well plates at a density of 1.5 × 10⁴ cells per well in quadruplets 24 h prior to stimulation. Cells were stimulated with 0.5 µg blocked sEV for 4 h at 37°C and Luciferase-assay was performed using Nano-Glo^® Luciferase (Promega, Madison, United States) according to the manufacturer`s instructions.

Hydrogen peroxide (H₂O₂) 35% was purchased from Sigma-Aldrich (349887, Merck KGaA, USA). Dulbecco’s modified Eagle’s medium (DMEM) and Ham’s F12 medium were obtained from GIBCO (Life Technologies; Carlsbad, USA). CellTiter-Glo assay (G7572), Nano-Glo luciferase and ROS-Glo Hydrogen Peroxide assay kits were purchase from Promega Corporation (Madison, WI, USA). Chloroquine (CQ; Sigma-Aldrich, C6628) and Concanavalin A (ConA, MERCK, MO, 344085) were dissolved in dimethyl sulfoxide (DMSO) to prepare stock solutions. Human RPE cell cultures (ARPE-19) were purchased from the American Type Culture Collection (CRL-2302; ATCC) and cultured as previously described [12 (link)].

The C. difficile complementation assay was carried out as described previously with modification (92 (link), 99 (link)). Briefly, C. difficile cells carrying CBD-BitLuc^opt construct were grown until OD₆₀₀ reached 0.4 to 0.5. The culture was induced with 4% (wt/vol) d-xylose for 2 h. To measure luciferase activity, 50 μL Nano-Glo Luciferase (Promega N1110) was mixed with 50 μL of OD₆₀₀ 0.2 normalized C. difficile culture, according to the manufacturer’s instructions. Measurements were performed in triplicate in a 96-well white flat-bottom plate (Greiner bio-one 655073). The luminescence signal was measured using a Hidex sense microplate reader. Statistical analysis was performed by using Prism 9 (GraphPad, Inc, La Jolla, CA). Statistical significance was calculated using Student’s t tests.

For the NFκB reporter assay, 100,000 cells were seeded in a 96-well flat-bottom plate and serial dilution was performed for antibodies. After 24 h, luciferase activity was quantified using BriteLite Plus (catalog no. 6066761, PerkinElmer) and measured using an EnSpire Alpha multimode plate reader (catalog no. 2300, PerkinElmer). Jurkat-NFκB-GITR⁺ stable cells were generated by transduction using lentivirus particles. Transduced cells were sorted and screened for human GITR and NFκB expression, then incubated with anti-CD3 for 48 h to induce upregulation of PD-1 expression, which was later confirmed by flow cytometry. Next, a serial dilution of anti-huPD-1-huGITR-L was added to cells and luciferase activity was evaluated using Nano-Glo Luciferase (catalog no. N1120, Promega). Cells were transferred to flat-bottom plates and substrate was added before measurement using an EnSpire Alpha multimode reader. For the NFAT reporter assay, 4 × 10⁵ PD-L1-expressing CHO K1 activator cells were plated on 96-well flat-bottom plates and incubated overnight at 37 °C with a serial dilution of antibodies and 40 µl of 1.25 × 10⁶ cells ml^–1 human PD-1 NFAT reporter Jurkat cells. After 6 h, 80 µl of Bio-Glo reagent was added to each well and plates were incubated for 5 min at ambient temperature. Luminescence was measured as previously described.