Firefly luciferase

A total of 20,000 HEK293T cells were plated into each well of a poly-l-lysine–coated 96-well flat-bottom plate and allowed to adhere overnight. A constitutive cytokine receptor vector (2.5 ng), a STAT response element vector that drives Firefly luciferase (100 ng; Promega, Madison, WI) and Renilla luciferase control reporter vector (1 ng; Promega, Madison, WI) were mixed in a final volume of 5 μl in Opti-MEM (Gibco, Thermo Fisher Scientific, Waltham, MA) (“DNA mix”). Lipofectamine 2000 (0.3 μl; Invitrogen, Thermo Fisher Scientific, Waltham, MA) in 5 μl of Opti-MEM was incubated at room temperature for 5 min and then added to the DNA mix. The mixture was incubated at room temperature for 20 min, and the total volume of 10 μl was added to each well containing HEK293T cells. Twenty-four hours after transfection, cells were left untreated, treated with AP1903 (1 μg/ml; APExBio, Houston, TX), or with the indicated cytokines diluted in serum-free media. At the indicated time points after treatment, reporter activity was determined using the Dual-Glo Luciferase Assay System (Promega, Madison, WI) as per the manufacturer’s instructions. Firefly luciferase activity was first normalized to Renilla luciferase activity, and then STATresponse element fold induction was calculated by normalizing to untransfected controls.

Add 20 μL of cells to the tube, add 100 μL of firefly luciferase (Promega, USA) detection reagent, mix well and use the measured value and record. Then 100 μL of renilla luciferase detection working solution (Promega, USA) was added, and the value was detected and recorded. Relative luciferase activity (Firefly LUC/Renilla LUC) was obtained and the experiment was repeated three times.

The test antibodies were incubated with HSV-infected Vero cells or HEK293T cells stably expressing gB and engineered Jurkat reporter cells stably expressing the FcγRIIIa receptor, V158 (high affinity) variant, and an NFAT response element driving expression of firefly luciferase (Promega). Vero cells were infected with HSV-1F/2G at MOI 1. Twenty hours after infection, cells were harvested and distributed in white flat bottom 96 well plates (1.25 × 10⁴ cells per well) and incubated 6 h together with Jurkat effector cells at an effector:target ratio of 6:1 and serial dilutions of test antibodies. Noninfected Vero cells and Raji cells incubated with Rituximab served as negative and positive controls, respectively. Luciferase substrate was added and after 15 min incubation luminescence intensity was measured using a plate reader. Triplicate reads were performed, and means were calculated. Plate Background from control wells was subtracted. Fold of ADCC induction was calculated.

Kinetic data of luminescence production in full length firefly luciferase was obtained under the same conditions as that in FLCA. Briefly, firefly luciferase was purchased from Promega (Wisconsin, USA). Luciferase enzyme (150 nM or 450 nM) was suspended in 100 mM MOPS, 19 mM MgSO₄, pH 7.3. One microliter of the enzyme solution and 50 μL of a 2x ATP solution (40 mM ATP in 100 mM MOPS, 10 mM MgSO₄, pH 7.3) was dispensed to a well in a white 96-well plate (Corning-Costar, NY, USA). The luminescence was measured immediately after injection of a 2x LH₂ solution (150 μM LH₂, 100 mM MOPS, 10 mM MgSO₄, pH 7.3) with periodical integration for 0.2 s for 120 s using Synergy 2 luminometer (Biotek, Vermont, USA).

PCR was used to amplify the c-myc core promoter from HeLa genomic DNA using the c-Myc promoter primer as shown in Table 1. PCR conditions are described in Supporting information: S1 Table. The 468 bp PCR product was purified using a HiYield^™ Gel/PCR DNA Fragments Extraction Kit (RBC Bioscience, Taipei, Taiwan) and cloned into pGEM-T vector (Promega, Madison, WI, USA). The constructed plasmid was transformed into Escherichia coli (E. coli) strain DH5α. The product containing c-myc core promoter in pGEM-T vector was subcloned into the pGL3-Basic vector, which lacks eukaryotic promoter sequences and contains the firefly luciferase (Promega) as a reporter. The c-myc core promoter sequence was confirmed by sequencing analysis.