One glo luciferase assay reagent

HeLa cells (7 × 10⁴) were cotransfected in triplicate in a 96-well plate with 50 ng each of pGL4-BST2pro, lenti dCAS-VP64_Blast, lenti MS2-P65-HSF1_Hygro, and 100 ng of either pLV-sgR-BST2pro-Zeo or an empty vector control using the FuGENE6 transfection reagent (Promega) according to the manufacturer’s instructions. After 48 h, the cells were lysed in 100 μl of One-Glo Luciferase Assay Reagent (Promega). The firefly luciferase activity was determined with a Centro LB960 (Berthold) luminometer.

FL-melanoma and ISRE/FL-melanoma cells were seeded into 12 well plates and incubated at 37°C in 5% CO₂. Supernatants from either EGFP-ADSCs or IFNγ-ADSCs were added to these cells once they reached cell densities of 10⁵ and 3 × 10⁵ respectively. Two days later, a volume of ONE-Glo™ luciferase assay reagent (Promega Corp. Madison, WI) equal to that of the CM was added to each well and samples were mixed thoroughly. Luminescence imaging was performed in an imaging system (Bruker Inc, Ettlingen, Germany) using a UV-Epi-Illumination source with a 15 min exposure time.

HEK cells stably expressing firefly luciferase under the control of the human IFNB promoter (p125-HEK cells) have been described previously²³ . Cells were seeded at 20,000 cells per well in a 96-well plate. The following day, cells were transfected with the indicated doses of RNA extracted from the lungs of mice complexed with 0.5 μl Lipofectamine 2000 (Thermo Fisher Scientific) per well. Activation of the IFNB promoter was assessed after 24 h using OneGlo luciferase assay reagent (Promega).

To generate luciferase reporter viruses pseudotyped with either VSV-G or HIV-1-Env, 2.5 × 10⁵ 293T cells were transfected with either increasing amounts (0, 60, or 120 ng) of mammalian MARCH8 expression plasmids (WT, AxxL, and RING-CH mutants) or fixed amounts (100 ng) of MARCH8/MARCH3-chimeric expression plasmids, along with 20 ng of pC-VSVg or pC-NLenv, 500 ng of pNL-Luc2-IN/HiBiT-E(−)Fin, and an empty vector to reach a total DNA amount of 1 μg, using FuGENE6. Sixteen hours post-transfection, the cells were washed with PBS and incubated with 1 mL of fresh complete medium. After an additional 24 h, the supernatants containing the pseudotyped viruses were treated with 37.5 U/ mL of DNaseI (Roche Applied Science, Penzberg, Upper Bavaria, Germany) at 37 °C for 30 min. Viral supernatants were quantified using the HiBiT assay, as described previously [20 (link)]. The HiBiT-based luciferase activity in the viral supernatants was measured using a Centro LB960 luminometer (Berthold, Bad Wildbad, Baden-Württemberg, Germany) and converted into p24 antigen levels. To assess viral infectivity, 1 × 10⁴ MAGIC5 cells were exposed to 1 ng of p24 antigen from the HIV-1 supernatants. After 48 h, the cells were lysed in 100 μL of One-Glo luciferase assay reagent (Promega), and firefly luciferase activity was quantified using a Centro LB960 luminometer.

MTT [3- (4, 5-dimethylthiazol-2-yl)-2, 5-dipheny ltetrazolium bromide] was purchased from Alfa Aesar, Inc. (Lancashire, United Kingdom), reconstituted in PBS to 2 mg/mL and diluted with appropriate cell culture media before use. Lipofectamine 3000^®, TNFα and ON-TARGETplus^® non-targeting siRNAs were obtained from Life Technologies Inc (Carlsbad, CA, USA), Peprotech, (Rocky Hill, NJ, USA) and GE Dharmacon^® (Little Chalfont, Buckinghamshire, UK) respectively. Annexin V-FITC apoptosis detection kit and Cellytic M^® buffer were from Sigma-Aldrich (St. Louis, MO, USA). Pierce transcription factor assay kits for NF-κB p65/p50 and NE-PER^® nuclear and cytoplasmic extraction reagents were from Thermo Scientific (Rockford, IL, USA). All antibodies were from Cell Signaling Technology Inc. (Danvers, MA, USA) except for anti-GADPH and anti-p50 (Ser337) antibody which were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Product codes of primary antibodies are as follows: cleaved caspase-3 (#9664); Bcl-2 (#2872); Mcl1 (#4572); Bcl-xl (#BAX (#2772); survivin (#2803); p65 (#8242); p65 Ser536 (#3031); p105/50 (#3035); p50 Ser337 (sc-33022); IκBα (#4812); GAPDH (sc-48166). p50 or p65 siRNA were also from Santa Cruz. ONE-Glo™ Luciferase assay reagent was from Promega (Madison, WI, USA).