The core-defective, assembly deficient single-cycle infectious HCV (HCVsc) was generated from a trans-packaging system as previously described (Masaki et al. 2010 (link); Li et al. 2014 (link)). HCVsc is able to enter and replicate viral RNA in hepatocytes but unable to produce progeny viruses, thus recapitulating only the early stages of the HCV life cycle (entry through genome replication), but not the late stages (virion assembly and secretion). In the assay, Huh7.5.1 cells were treated with various miRNA mimics at 25 nmol/L for 72 h in 96-well white plates (in 5 replicates) before infection with HCVsc. At 48 h post-infection, cells were harvested and measured for firefly luciferase activities per manufacturer’s instructions (Promega) using a POLARstar multidetection microplate reader (BMG Labtech).
Firefly luciferase activities
Manufactured by Promega
Sourced in United States
Firefly luciferase activities is a lab equipment product that measures the activity of firefly luciferase, an enzyme responsible for the bioluminescence in fireflies. The product provides a means to quantify the amount of firefly luciferase present in a sample.
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Lab products found in correlation
4 protocols using firefly luciferase activities
1
HCV Single-Cycle Infection Assay
2
HCV and VSV Pseudotyped Particle Assay
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HCVpp and VSVpp were produced as described previously [28 (link),29 (link)] with plasmids kindly provided by B. Bartosch and F.L. Cosset (Lyon, France). Pseudotyped particles were inoculated on 293T, 786-O or Huh-7 cells for 3 h at 37°C. At 72 h post-infection, cells were lysed and processed to measure the Firefly luciferase activities as indicated by the manufacturer (Promega). Luciferase activities with HCVpp were normalized for luciferase activities obtained with VSVpp. In each figure, results are reported as the mean ± S.D. of at least three independent experiments.
3
Bcl6 Transcriptional Regulation Assay
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To directly detect the regulatory effect of Bcl6 on the targeted genes, the 5′ UTR of the genes were amplified from genomic DNA and cloned in PGL3 vector. The binding sequences-mutant plasmids were generated by PCR-based site-directed mutagenesis. The reporter plasmids and pEGFP-Bcl6 vector were then transfected into RAW264.7 cells using Jet-ENDO transfection reagents (Polyplus, Illkirch, France). 6 h and 24 h later, the cells were collected and assayed for firefly luciferase activities (Promega, USA) with or without LCM stimulation.
4
Npt2a Promoter-Luciferase Reporter Assay
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Npt2a promoter-luciferase reporter gene constructs (NpT2a-luc) containing -208 and -4,400 bp were a generous gift of Heine Murer [6] . Npt2a-luc or pGL3-basic and were transfected into OK or OK-H cells using Lipofectamine 2000 (Invitrogen). At 24 h posttransfection, firefly luciferase activities were determined (Promega) according to the manufacturer's protocol and normalized to protein. The cells were transfected in triplicate and luciferase:protein ratios were determined per well and the average values determined per experiment. Three independent experiments were performed with each independent experiment using different cell culture stocks. In a fourth independent experiment, cells were co-transfected in triplicate with the Npt2a-luc reporter constructs and pSV-β-galactosidase vector (Promega) and the average luciferase:βgal activity ratios determined (Promega).
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