Production and Collection of Pseudovirus Particles Using Mammalian Cell Culture

Samenvatting

Source: Jamieson, T. R., et al. Detection of SARS-CoV-2 Neutralizing Antibodies using High-Throughput Fluorescent Imaging of Pseudovirus Infection. J. Vis. Exp. (2021)

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.

Protocol

1. VSV-S-EGFP pseudovirus preparation
   1. Infection
      1. 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’s modified eagle medium) for 1 h at 37 °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 °C with 5% CO₂.
         NOTE: A temperature of 34 °C is required for the propagation of VSV-S-eGFP in cell culture.
      2. 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.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
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