Production and Collection of Pseudovirus Particles Using Mammalian Cell Culture

Begin with a culture plate containing a monolayer of adherent mammalian cells.

Take engineered pseudovirus particles diluted in serum-free media.

The virus contains coronavirus spike proteins and an RNA encoding green fluorescent protein (GFP).

Add the virus to the monolayer and incubate with occasional rocking for viral attachment.

Replace the media with serum-containing media and incubate again.

Viral spike proteins bind to host cell receptors, enabling viral entry and RNA release into the cytoplasm.

The RNA replicates and synthesizes viral proteins, including GFP. New viral particles assemble, exit the cell, and spread to infect neighbouring cells.

Observe the cells under a fluorescence microscope.

Virus-infected cells show green fluorescence.

Over time, infected cells get damaged and detach from the surface, leaving gaps in the monolayer while retaining fluorescence.

Transfer the supernatant to a tube and centrifuge to remove cell debris.

Collect the supernatant containing pseudovirus particles.

To prepare the VSV spike EGFP pseudovirus for titering, infect the cells at 0.01 multiplicity of infection with the stock virus diluted in 12 milliliters of serum-free DMEM.

After adding the virus, incubate the cells for one hour at 37 degrees Celsius and 5% carbon dioxide with occasional rocking. At the end of the incubation, replace the inoculum with fresh DMEM supplemented with 2% FBS and 20 millimolar HEPES, then incubate the cells at 34 degrees Celsius and 5% carbon dioxide for 24 hours. The next day, use a fluorescent microscope to visualize the EGFP expression of the infected cells.

Once an extensive cytopathic effect and cell detachment are observed, collect the culture supernatant and remove the debris by centrifugation.