Sample Preparation for Luciferase-Based Viral Quantification

Begin with a multiwell plate containing epithelial cells infected with a genetically modified virus.

The virus carries a luciferase enzyme-encoding transgene.

Take fresh cultures of epithelial cells in a white-walled multi-well plate.

Transfer the supernatant containing unknown concentrations of virus into the white-walled wells.

Simultaneously, add known concentrations of the virus as standards.

Centrifuge the plate to promote close contact between the viral particles and the host cell surface receptors, and incubate.

The virus enters the host cell and delivers its negative-strand RNA genome.

The viral RNA polymerase then transcribes the genome into mRNA.

Using the host’s cellular machinery, the mRNA is translated into viral proteins, including luciferase.

The amount of luciferase accumulated inside the host cell increases with increasing viral concentration.

The samples are ready for further estimation of viral concentrations using the luciferase assay.

Prepare a standard curve of VSV-Delta-51 in serum free DMEM such that the final concentration of plaque forming units per milliliter after transfer onto Vero cells is as follows. Prepare 50 microliters of each concentration per plate of Vero