Determining Optimal Target Cell Density Using a Luciferase-Based Pseudovirus Infection Assay

Begin with a multi-well plate containing adherent mammalian cells at increasing densities.
Add an equal concentration of pseudovirus to the wells and incubate.
The pseudovirus bears influenza envelope proteins on a lentiviral core and contains a gene encoding luciferase.
The virus binds to cell surface receptors and undergoes endocytosis.
Once inside, the viral RNA is released and reverse-transcribed into DNA by reverse transcriptase. The DNA then integrates into the host genome, resulting in luciferase expression.
Remove the culture medium and wash the cells with buffer.
Add lysis buffer and incubate at a low temperature. The lytic reagent disrupts cells and releases luciferase.
Let the lysates reach room temperature and transfer them to an opaque multi-well plate.
Add luciferase substrate. Luciferase catalyzes substrate oxidation, producing luminescence.
Using a luminometer, measure luminescence to quantify viral infection.
Compare luminescence levels across cell densities. The highest luminescence indicates the optimal density with the highest infection efficiency.

For pseudovirus titration, make a single-cell suspension of five times 10 to the fourth cells per milliliter of MDCK cells in a complete DMEM medium.

Add 1,250, 2,500, 5,000, 10,000, 20,000, and 40,000 cells to different wells of a flat-bottom 96-well plate. After incubation, thaw and vortex the pseudovirus. Add six HAU of pseudo viruses to each well of a 96-well round-bottom plate and replenish each well with complete DMEM up to 120 microliters.

Transfer 100 microliters of the mixture to the cells in the 96-well plate. Incubate the culture plate for 48, 60, and 72 hours for viral infection and perform a luciferase assay. 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 pseudovirus by using the luminometer.