<ol><li>VSV-S-EGFP pseudovirus preparation<ol style="list-style-type:decimal;"><li>Infection<ol style="list-style-type:decimal;"><li>Infect cells at multiplicity of infection (MOI) 0.01 with VSV-S-eGFP (vesicular stomatitis virus - spike protein - enhanced green fluorescent protein) stock virus in 12 mL of serum-free DMEM (Dulbecco&#x2019;s modified eagle medium) for 1 h at 37 &#xB0;C with 5% CO2, occasionally rocking the plates. Replace the inoculum with fresh DMEM (containing 2% FBS or fetal bovine serum and 20 mM HEPES, or 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.7), and move to an incubator set to 34 &#xB0;C with 5% CO2. NOTE: A temperature of 34 &#xB0;C is required for the propagation of VSV-S-eGFP in cell culture.</li><li>Collect cell supernatants upon observation of extensive cytopathic effect (CPE) and cell detachment, approximately 48 h post-infection. Use a fluorescent microscope to visualize the extensive expression of eGFP by infected cells starting at 24 h post-infection.</li></ol></li></ol></li></ol>

<table class="ck-table-resized"><tbody><tr><td>Dulbecco&#x27;s modified Eagle&#x27;s medium (Gibco)</td><td>Fisher scientific</td><td>10-013-CV</td><td>&#xA0;</td></tr><tr><td>Dulbecco&#x2019;s Phosphate-Buffered Saline (DPBS)</td><td>Fisher scientific</td><td>21-031-CV</td><td>&#xA0;</td></tr><tr><td>4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)</td><td>Fisher scientific</td><td>BP-310-500</td><td>&#xA0;</td></tr><tr><td>Vero E6 cells</td><td>ATCC</td><td>CRL-1586</td><td>&#xA0;</td></tr></tbody></table>

production and collection of pseudovirus particles using mammalian cell culture

Source: <a href="https://app.jove.com/v/62486/detection-sars-cov-2-neutralizing-antibodies-using-high-throughput">Jamieson, T. R., et al. Detection of SARS-CoV-2 Neutralizing Antibodies using High-Throughput Fluorescent Imaging of Pseudovirus Infection. J. Vis. Exp. (2021)</a>This video demonstrates the procedure for infecting mammalian cells with green fluorescent protein (GFP)-encoding pseudovirus, monitoring viral replication and spread by fluorescence microscopy, and collecting virus-containing supernatant for downstream applications.

Production and Collection of Pseudovirus Particles Using Mammalian Cell Culture

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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.

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83 Views. 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 e...

Production and Collection of Pseudovirus Particles Using Mammalian Cell Culture

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Name	Company	Catalog Number
Dulbecco's modified Eagle's medium (Gibco)	Fisher scientific	10-013-CV
Dulbecco’s Phosphate-Buffered Saline (DPBS)	Fisher scientific	21-031-CV
4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)	Fisher scientific	BP-310-500
Vero E6 cells	ATCC	CRL-1586