Source: Yadavalli, T., et al. Porcine Corneal Tissue Explant to Study the Efficacy of Herpes Simplex Virus-1 Antivirals. J. Vis. Exp. (2021)
This video demonstrates the method to perform a plaque assay using a virus obtained from infected porcine cornea. The porcine cornea is used to test the antiviral efficacy of experimental drugs.
1. Virus infection quantification
NOTE: Virus titers from porcine corneas should be evaluated every day to analyze the effect of drug treatment.
2. Virus quantification by Plaque assay
NOTE: To perform a plaque assay, grow and plate Vero cells into a 6-well plate and ensure 90% confluency of cells before the start of the assay. Use a confluent 75 cm2 flask of these cells. All the steps below need to be performed inside a biosafety cabinet.
The authors have nothing to disclose.
30 G hypodermic needles. | BD | 305128 | |
500 mL glass bottle. | Thomas Scientific | 844027 | |
Biosafety cabinet with a Bio-Safety Level-2 (BSL-2) certification. | Thermofisher Scientific | Herasafe 2030i | |
Calgiswab 6" Sterile Calcium Alginate Standard Swabs. | Puritan | 22029501 | |
Cell scraper – 25 cm | Biologix BE | 70-1180 70-1250 | |
centrifuge tube | |||
Crystal violet | Sigma Aldrich | C6158 | Store the powder in a dark place |
Dulbecco's modified Eagle's medium – DMEM | GIBCO | 41966029 | Store at 4 °C until use |
Ethanol | Sigma Aldrich | E7023 | |
Fetal bovine serum -FBS | Sigma Aldrich | F2442 | Aliquot into 50 mL tubes and keep frozen until use |
Vero cells | American Type Culture Collection ATCC | CRL-1586 | |
Vortex machine | |||
Methanol | |||
Methylcellulose | |||
Microcentrifuge tube | |||
Porcine Corneas | Park Packaging Co., Chicago, IL | Special order by request | |
Procedure bench covers – as needed. | Thermofisher Scientific | S42400 | |
Serological Pipettes | Thomas Scientific | P7132, P7127, P7128, P7129, P7137 | |
Serological Pipetting equipment. | Thomas Scientific | Ezpette Pro | |
Stereoscope | Carl Zeiss | SteREO Discovery V20 | |
Tissue culture flasks, T175 cm2. | Thomas Scientific | T1275 | |
Tissue culture incubators which can maintain 5% CO2 and 37 °C temperature | Thermofisher Scientific | Forma 50145523 | |
Tissue culture treated plates (6-well). | Thomas Scientific | T1006 | |
Trypsin |