Bright glo luciferase assay reagent

To assess the antibody-dependent cytotoxicity of anti-MERS antibodies, an FcγRIIIa reporter assay was performed and optimized. Jurkat/NFAT-luc/FcγRIIIa effector cells were developed as previously described^{40 ,41 (link)}. The target 293T/MERS cells and the effector Jurkat/NFAT-luc/FcγRIIIa cells were counted and diluted in the dilution medium (RPMI 1640 with no phenol red and with 2% low IgG FBS) and were seeded into white 96-well plates (2.5 × 10⁴ 293T/MERS cells/well in 25 μL of dilution medium and 1 × 10⁵ Jurkat/NFAT-luc/FcγRIIIa cells/well in 25 μL of dilution medium, and the ratio of the effector to the target cell was 4:1). Anti-MRES antibodies were serially diluted in a 1:2.5 ratio with dilution medium at an initial concentration of 2 μg/mL, and 50 μL of the serial antibody diluents were added into the 96-well plates. Cultures were incubated at 37 °C with 5% CO₂ for 16 h. By adding of Bright-Glo Luciferase Assay reagent (Promega) and measuring relative luciferase units (RLU) with a plate reader (SpectraMax), the ADCC activity of anti-MERS antibodies can be determined. In this study, the ADCC reporter gene assays that have not been described specifically refer to this method.

293T cells (obtained from the American Type Culture Collection (ATCC)) were co-transfected with 400 ng of APOBEC3 plasmid or an empty expression vector, 600 ng of proviral HIV-1 plasmid (HIV-1ΔVifΔEnvLuc2) with an SIV vif, and 200 ng of L-VSV-G for pseudotyping, using TransIT-LT1 (Mirus Bio). The cells and the virus supernatant were collected 48–72h post transfection. The harvested cells were used for the western-blot analyses and the following antibodies were used: mouse HA-specific antibody (Balco), mouse anti-tubulin and anti-actin antibodies (Sigma-Aldrich), and secondary goat anti-mouse horseradish peroxidase-conjugated antibody (Sigma-Aldrich). The total amount of virus in the supernatant was quantified by p24 Gag ELISA assay (Advanced Bioscience Laboratories). Each transfection condition was performed in 2–3 independent experiments. For the infection, SupT1 cells (obtained from the NIH AIDS Repository) were plated at 0.4 M cells/ml with 20 μg/ml of diethylminoethyl-dextran and infected with 2 ng of virus. Infections were performed in triplicate and luciferase activity was measured after 72h with the Bright-Glo Luciferase Assay Reagent (Promega).

The predicted open reading frame (ORF) for candidate GPCR genes were cloned into pCDNA3.1 expression vector (Invitrogen, Carlsbad, CA). Human embryonic kidney (HEK) 293 cells were cultured in DMEM medium supplied with 10% FBS at 37 °C with 5% CO₂. Cells were plated onto a white opaque 96-well plate (2~5 × 10⁴ per well) 8 hours prior to transfection. Cells were then transiently transfected with pCDNA3.1/GPCR constructs together with a human Gα15 expression construct and a 6 × CRE-luciferase reporter construct (Kindly provided by Dr. Alan S. Kopin, Molecular Pharmacology Research Center, Tupper Research Institute, Tufts–New England Medical Center) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Thirty-six hours post-transfection, cells were exposed to ligands (JH III or DA, dopamine hydrochloride, Sigma, St. Louis, MO) and 1 μM of forskolin for 4 hr in serum-free medium. Cells then were lysed using Bright-Glo™ Luciferase Assay reagent (Promega Corporation, Madison, WI) for 2 min in the dark. The luciferase activity was quantified using a SpectraMax M5 microplate reader (Molecular Devices, Sunnyvale, CA, USA). At least six replicates were used for each treatment.

To generate target cells expressing luciferase, lentiviral luciferase was transduced into NALM-6. In 96-well plates, CAR NK-92, CAR KHYG-1, or CAR T cells were co-cultured with luciferase-expressing NALM-6 at indicated E:T ratio and incubation time. At the end of incubation time, the Bright-Glo Luciferase assay reagent (E2650; Promega) was added to each well containing effector/target cell mixture. After shaking the plate for 5 min at room temperature, luminescence was detected using an EnVision reader (PerkinElmer).

Stable ptkGC3p53luc-bsd SiHa, CaSki, and HeLa cell lines as well as the composition of the p53 reporter plasmid ptkGC3p53-luc have been described earlier [17] (link). The cells were seeded into 96-well plates (10⁴ cells/well). After allowing for cell attachment for 24 hours, the treatments were begun for the indicated durations. The living cells in each well were determined colorimetrically with the WST-1 assay. Thereafter, the cells were rinsed with PBS and overlaid with 100 µl of a mixture containing 50% PBS and 50% Bright-Glo luciferase assay reagent (Promega, Madison, WI, USA). The luciferase activity was quantified with the aid of a hybrid capture luminometer (Digene, Gaithersburg, MD, USA). Luciferase readings were divided by WST-1 value to obtain normalized reporter activity.

The sera were diluted, transferred into 96-well white flat-bottom culture plates, and then mixed with the SARS-CoV-2 pseudoviruses (1 × 10⁵ TCID50/well). After 30 mins incubation at 37°C, the trypsinized BHK-21-hACE2 cells were added into the 96-well white flat-bottom culture plates with viruses/serum samples at a density of 2 × 10⁴/well. After 16 h, the medium of the infected cells was removed and lysed by 1 × Bright-Glo Luciferase Assay reagent (Promega) for at least 2 mins. Luciferase activity was detected with a SpectraMax iD3 Multi-well Luminometer (Molecular devices) within 5 mins. The 50% neutralization dilution titer (NT50) was calculated by GraphPad Prism 8 software with nonlinear regression curve fitting (normalized response, variable slope).