To measure replication of the Gaussia luciferase reporter HCV, we used the Gaussia Glow Assay kit (Thermo Scientific, Waltham, MA, USA). Briefly, 30 μL of cell supernatant was added to 30 μL of Luciferase Cell Lysis Buffer (Thermo Scientific, Waltham, MA, USA) in black 96-well microplates and incubated at room temperature for 20 minutes. About 50 μL of 1× Coelenterazine was added according to the manufacturer’s instructions, and luciferase activity (relative light units (RLU)) was measured with an EnSpire 2300 Multilabel Plate Reader (PerkinElmer, Duluth, GA, USA), and normalized to an uninfected control.
Gaussia glow assay kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Gaussia Glow Assay kit is a bioluminescent detection system for monitoring gene expression and cellular events. The kit utilizes the Gaussia luciferase reporter protein, which emits light upon the addition of the substrate coelenterazine. This light output can be detected using a luminometer, providing a quantitative measure of the reporter activity.
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2 protocols using gaussia glow assay kit
1
Quantifying HCV Replication with Gaussia Luciferase
2
Evaluating Gluc reporter activity with miRNA
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For Gluc reporter assays, transfections were performed with Lipofectamine 3000 (Thermo Fisher Scientific) in a 24 well plate according to manufacturer’s recommendations. Cells were transfected with 500 ng of either CMV-Gluc, CMV-Gluc-miR122a*3, CMV-Gluc-miR199a*3 or CMV-Gluc-miR122a*3-miR199a*3 and the amount of secreted Gluc was quantified after 48–72 hours using the Gaussia glow assay kit (Thermo Fisher Scientific). The chemiluminescence was measured in the Infinite 200 Pro NanoQuant (Tecan Trading AG, Switzerland). Gluc expression after transfection of cells with CMV-Gluc-miR122a*3, CMV-Gluc-miR199a*3, or CMV-Gluc-miR122a*3-miR199a*3 was normalized with Gluc expression after transfection with CMV-Gluc and reported as its percentage.
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