This video describes the virus plaque assay using coronavirus pseudovirus particles. The diluted pseudovirus is added to a monolayer of mammalian cells, followed by incubation with a methylcellulose overlay. Plaques formed by infected cells are counted visually to determine the infectious virus titer.
Method Article
Quantification of Pseudovirus Particles by Plaque Assay
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September 30th, 2026
In This Article
Abstract
Protocol
Titering the vesicular stomatitis virus expressing coronavirus spike protein and enhanced green fluorescent protein (VSV-S-eGFP) pseudovirus
1. Serial dilution to determine viral titer
1. Plate Vero E6 cells on 6-well plates at a seeding density of 6 × 105 cells per well in Dulbecco's Modified Eagle Medium or DMEM (with 10% FBS (Fetal Bovine Serum)), and incubate overnight at 37 °C with 5% CO₂.
NOTE: Vero cells that overexpress TMPRSS2 and hACE2 can also be used.
2. Set up a 10-fold serial dilution series of the VSV-SeGFP virus in cold serum-free DMEM by placing 900 μL of media in seven microcentrifuge tubes. Label tubes from 1 to 7. Add 100 μL of the viral stock to the first tube and vortex briefly. Transfer 100 μL of diluted virus from tube 1 to tube 2 and vortex briefly; continue until tube 7.
3. Aspirate the medium from all wells of the 6-well plate containing Vero E6 cells, and replace with 500 μL of dilutions 105 to 107. Incubate the plates for 45 min at 37 °C with 5% CO₂, gently rocking every 15 min.
4. Aspirate the inoculum and replace with overlay containing a 1:1 mixture of 2x DMEM and 6% carboxymethyl cellulose (CMC), final concentration of 3% CMC, supplemented with 10% FBS; incubate at 34 °C with 5% CO₂ for 48 h.
NOTE: Pre-warm the overlay mixture in a 37 °C water bath for 15 min during the infection step. A 1:1 mixture of 1% agarose with 2x DMEM supplemented with 10% FBS may be used in place of CMC. To overlay with agarose, boil 1% agarose (in dH₂O) and mix with cold 2x DMEM. Ensure the mixture is a suitable temperature before adding to the cell monolayer (approximately 37-40 °C). If agarose is used for the overlay, cells must be fixed by incubating in 2 mL of 3:1 methanol:acetic acid for at least one hour prior to staining.
2. Staining and calculating viral titer
1. Visualize plaques by staining with crystal violet or direct visualization of eGFP under a fluorescent microscope.
2. To stain plates, aspirate the overlay, wash once with PBS, then add 2 mL of 0.1% crystal violet (in 80% methanol and dH₂O) to each well. Place on a plate rocker for approximately 20 min at room temperature prior to de-staining. Remove the crystal violet, and gently wash each well twice using dH₂O or PBS.
NOTE: Washing and staining steps should be performed gently by pipetting slowly towards the side of each well to ensure the cell monolayer remains intact throughout the procedure.
3. Allow plates to dry for at least 1 h prior to counting plaques. Ensure that plaque counts are obtained from wells containing 20 to 200 plaques. To calculate the titer of the virus in plaque-forming units (PFU) per mL, multiply the dilution factor of the well counted with the volume of infection, and divide this number from the plaques present in the respective well.
Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| 0.25% trypsin-EDTA (Gibco) | Fisher scientific | LS25200114 | |
| Vero E6 cells | ATCC | CRL-1586 | |
| carboxymethyl cellulose | Sigma | C5678 | |
| Dulbecco's modified Eagle's medium (Gibco) | Fisher scientific | 10-013-CV | |
| Dulbecco's modified Eagle's medium (Powder) (Gibco) | Thermo Fisher Scientific | 12-800-017 | |
| Dulbecco’s Phosphate-Buffered Saline (DPBS) | Fisher scientific | 21-031-CV | |
| Penicillin/streptomycin | Thermo Fisher Scientific | 15070063 | |
| SARS-CoV-2 (2019-nCoV) Spike Neutralizing Antibody, Mouse Mab | SinoBiological | 40592-MM57 |
