Steady glo luciferase assay kit

Cells were seeded at 3 × 10⁴ cells/well in 48-well plates and allowed to proliferate until 60%–80% confluent. Complexes of phage vectors and transfection reagents prepared at optimal ratios in serum-free media, or control phage vector alone were applied to the cells, followed by incubation at 37 °C for 4 h. Next complete media, containing FBS, was administered and the cells were incubated at 37 °C to allow for transgene expression. Transduction efficiency of the phage complexes was determined by using phage carrying the green fluorescent protein (GFP) or firefly luciferase (Luc) reporter genes. GFP expression was evaluated using a Nikon eclipse TE200-S fluorescent microscope (Nikon, Surrey, UK). Luc reporter gene expression in transduced cells was determined by using the Promega Steady-Glo^® Luciferase Assay kit following the manufacturer’s protocol and quantified using a Promega plate reader (Promega, Southampton, UK). Data were normalized to 100 µg protein levels as determined by the Bradford assay and presented as relative luminescence units per 100 µg protein. Cell viability was analyzed by CellTiter-glo^® cell viability assay kit; following the manufacturer’s protocol and quantified using a Promega plate reader. All cell transduction experiments were repeated three times and performed in triplicates.

Virus stocks were prepared by transfecting HEK-293T cells with HIV-1 DNAs (3 μg). Cells were maintained for 24 h post-transfection with or without 5μM PF-46396, centrifuged and filtered to remove residual cell debris. The virus stocks were quantified for p24 antigen using HIV-1 p24 Antigen Capture kit (ABL, USA). Equal volumes (300 μl) of HIV-1 p24 normalized virus supernatants (5 ng HIV-1 p24) were used to infect TZM-bl cells (5 × 10⁴/well) in the presence of 20 μg DEAE-dextran per ml in 24 well plate. Single-round infectivity assays were performed as previously described45 (link). The luciferase activity in the cell lysates was measured using the Steady-Glo luciferase assay kit (Promega, USA) following manufacturer’s recommendations.

Luciferase activity was measured using the Steady-Glo Luciferase Assay Kit (Promega). 40 embryos, five wandering L3 larvae, five pupae or five female flies (2-day-old) were collected in 300 μL GLO lysis buffer for each sample; each luciferase assay contained five independent samples. Muscle and fat body of five L3 larvae, 20 brain discs, 40 wing discs, 40 gonads, 20 salivary glands of L3 larvae, and 10 guts from female flies were dissected in cold PBS and transferred to 90 μL GLO lysis buffer for each sample; three independent samples were used for each assay. Samples homogenized and then centrifuged at 12,000×g for 15 min, and then take 50 μL supernatant in 96-well plates. After incubation in the dark for 20 min, luminescence was measured on a luminometer (Thermo Scientific, VARIOSKAN FLASH).