Mar 18, 2020

Public workspaceSARS-CoV-2 virus plaque assays [Biosafety Level 3]

DOI
dx.doi.org/10.17504/protocols.io.bdtni6me
  • tenOever Lab1
  • 1Icahn School of Medicine at Mount Sinai
  • Coronavirus Method Development Community
Emma Ganley
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DOI: dx.doi.org/10.17504/protocols.io.bdtni6me
External link: https://bit.ly/3d6OUny
Protocol Citation: tenOever Lab 2020. SARS-CoV-2 virus plaque assays [Biosafety Level 3]. protocols.io https://dx.doi.org/10.17504/protocols.io.bdtni6me
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 18, 2020
Last Modified: March 18, 2020
Protocol Integer ID: 34382
Keywords: COVID19, SARS-CoV2, viral plaque assay,
Abstract
Method to plaque the SARS-CoV-2 virus




Image attribution:
Y tambe / CC BY-SA (http://creativecommons.org/licenses/by-sa/3.0/)
Downloaded from: https://commons.wikimedia.org/wiki/File:Plaque_assay_macro.jpg
Safety warnings
Biosafety Level 3
Media Recipes
Media Recipes
Media recipe for modified DMEM: [DMEM with 2% FBS]:

  • Amount475 mL DMEM (w/ L-Glutamate, Sodium pyruvate)
  • Amount5 mL 100x P/S
  • Amount5 mL 100x Non-Essential Amino Acids (NEAA) Solution
  • Amount5 mL 1M HEPES
  • Amount10 mL FBS

Media recipe for 2XMEM: Sterile filter with 22 μm filtration unit:

  • Amount100 mL 10x MEM
  • Amount10 mL 100x P/S
  • Amount10 mL 100x L-Glutamine
  • Amount6 mL 35%BSA
  • Amount340 mL sterile ddH2O
  • Amount10 mL 1M HEPES
  • Amount24 mL 5% NaHCO3
Overlay Media (Ensure this is just warm to the touch when added to cells)

  • Amount1 mL PBS
  • Amount31.5 mL 2X MEM
  • Amount17.5 mL Oxoid Agar

SARS-CoV2 virus plaque assays (Biosafety level 3)
SARS-CoV2 virus plaque assays (Biosafety level 3)
Seed Vero E6 cells in 6-well plates overnight at Temperature37 °C (106 cells/well)

Dilute the SARS-CoV-2 virus logarithmically in infection media
Replace media with modified DMEM and add diluted virus in each well (200 µl/well)

Allow virus adsorption for Duration01:00:00 , agitating the plate every Duration00:10:00 .

Heat the overlay media/oxoid agar mixture to ensure homogenous consistency and add it to each well (2ml/well), incubate at Temperature37 °C incubator for Duration72:00:00 .

Fix the plate with 5% formaldehyde (w/ methanol), (1.5 ml/per well) DurationOvernight to ensure virus inactivation and Stain with crystal violet for Duration01:00:00 to determine viral titers (PFU/ml)