JoVE Encyclopedia of Experiments
Microbiologia
0 vistas • 2:01 min • July 31st, 2026
Begin with a culture of epithelial cells infected with a pseudovirus, which is a genetically engineered non-virulent virus.
The pseudovirus carries a luciferase reporter gene that enables expression of the luciferase enzyme in the infected host cells.
Remove the media from the well.
Wash with a buffer to remove any residual media.
Add a lysis buffer to the well that aids in cell lysis.
Incubate at an ultra-low temperature to freeze the cells.
Thaw the cells at room temperature.
The freezing and thawing process ensures complete cell lysis, releasing intracellular components, including the luciferase enzyme.
Transfer the cell lysate to an opaque plate to reduce the background signal during luminescence measurement.
Add a substrate.
The luciferase enzyme in the lysate reacts with the substrate, emitting light.
Measure the emitted luminescence to quantify the pseudovirus infection level.
To perform the luciferase assay, wash the cells by removing the medium carefully and rinsing it with 200 microliters of PBS.
Remove as much of the PBS as possible, then add 50 milliliters of the lysis buffer to each well. On the next day, equilibrate the plates at room temperature for two hours, then rock the culture plates several times and transfer all the cell lysates to opaque 96 well plates. Add 50 microliters of the substrate to each plate and mix it well.
Record the luciferase titers of the pseudo virus by using the luminometer.