Apr 17, 2020
Viral Titration of SARS-COV-2 by Plaque Assay (Semi-Solid Agarose)
- Björn Meyer1
- 1Institut Pasteur
- Coronavirus Method Development Community
Protocol Citation: Björn Meyer 2020. Viral Titration of SARS-COV-2 by Plaque Assay (Semi-Solid Agarose). protocols.io https://dx.doi.org/10.17504/protocols.io.be4zjgx6
Manuscript citation:
Gordon, D.E., Jang, G.M., Bouhaddou, M.et al.A SARS-CoV-2 protein interaction map reveals targets for drug repurposing.Nature(2020). https://doi.org/10.1038/s41586-020-2286-9
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: April 14, 2020
Last Modified: April 17, 2020
Protocol Integer ID: 35705
Keywords: plaque assay, viral titration, Covid-19, Covid 19, Coronavirus,
Abstract
This protocol outlines the process of plaque assay for the viral titration of SARS-CoV-2.
Attachments
Materials
MATERIALS
Ethyl AlcoholMerck MilliporeSigma (Sigma-Aldrich)Catalog #E7023
MEMThermo FisherCatalog #11095080
UltraPure™ AgaroseThermo FisherCatalog #16500500
DMEM, high glucose, GlutaMAX™ Supplement, pyruvateThermo FisherCatalog #31966047
Gibco™ Trypsin-EDTA (0.05%) phenol redFisher ScientificCatalog #11580626
Gibco™ Fetal Bovine Serum qualified One Shot™ formatFisher ScientificCatalog #A3160802
Corning™ Costar™ Flat Bottom Cell Culture Plates (24 well)Fisher ScientificCatalog #10732552
Corning™ Costar™ Clear Polystyrene 96-Well Microplates 330µL With LidFisher ScientificCatalog #10360691
Formalin solution neutral buffered 10%Merck MilliporeSigma (Sigma-Aldrich)Catalog #HT501128-4L
Crystal violet solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #HT90132
VERO C1008 [Vero 76 clone E6 Vero E6] (ATCC® CRL-1586™)ATCCCatalog #CRL-1586
| Item | Reference | Storage conditions | |
| Agarose (Thermofisher) | 16500500 | RT | |
| DMEM (Thermofisher) | 31966047 | 4°C | |
| Trypsin (Gibco) | 11580626 | 4°C | |
| MEM (Thermofisher) | 11095080 | 4°C | |
| Fetal Bovine Serum (Thermofisher) | A3160802 | -20°C | |
| 24 well plates (Thermofisher) | 10732552 | RT | |
| 96 well plates round bottom (Thermofisher) | 10360691 | RT | |
| ETHANOL ABSOLUTE EXTRA PURE | 24103-5L-R | RT | |
| FORMALIN SOLUTION, NEUTRAL BUFFERED, 10% | HT501128-4L | RT | |
| CRYSTAL VIOLET SOLUTION | HT90132-1L | RT |
Safety warnings
Please refer to the Safety Data Sheets (SDS) for health and environmental hazards.
Before start
- Wear gloves during the entirety of the procedure.
- Use filtered tips only.
- Change tips as many times as possible.
- Decontaminate all tips and pipettes using a solution of diluted bleach.
Preparing Plaque Overlay (2x)
Preparing Plaque Overlay (2x)
Mix 455 mL MEM with 20 mL FBS .
Note
The final concentration will be 4% in the 2x overlay.
Prewarm the media to 37 °C .
Dissolve 0.6 g Agarose in 30 mL H2O .
Note
The final concentration will be 2% in the 2x overlay
Melt Agarose in the microwave until liquified.
Quickly add 25 mL melted Agarose to the prewarmed media from Step 2.
Close the bottle containing the Agarose/media mixture and shake vigorously for 00:00:30 , then again for 3-4 more time over the 00:10:00 time period.
Let the solution cool at Room temperature .
Note
Solution can be stored at 4 °C and can be reused when needed.
Day 1
Day 1
Plate 24 well plates with 7.5x104 Vero E6 per well in 10% FBS/DMEM.
Let cells grow Overnight at 37 °C .
Day 2 - Viral Dilutions
Day 2 - Viral Dilutions
Set up a 96-well plate in order to dilute your viral solution (serial dilutions).
In each well, put 270 µL MEM (serum-free) .
Add 30 µL viral solution in the first row (A) and mix thoroughly.
Note
Steps 11 to 22 are to be performed in a BSL-3 laboratory/setting.
Discard your tips.
Transfer 30 µL previous mix (solution in row A) to the second row (B).
Repeat that step from row C until row H (SARS-CoV-2 often reaches titers to 106).
Day 2 - Viral Infection
Day 2 - Viral Infection
Transfer 250 µL each dilution into each well .
Incubate at 37 °C for 01:00:00 . Every 00:15:00 , rock the plate a bit.
While cells are incubating with virus, prewarm 2x overlay media.
After 1 hour absorption time, add 250 µL 2x overlay media on top of the 250uL of inoculum (final volume 500uL per well).
Incubate at 37 °C for ~65:00:00 .
Day 5
Day 5
Prepare a solution of crystal violet diluted in EtOH as follows: 100 mL crystal violey + 200 mL EtOH + 700 mL water .
Put about 00500 µL formalin 4% into each well on top of the overlay media.
Let the well-plate sit for 00:20:00 to 00:30:00 at Room temperature .
Remove media from the well plate.
Add a solution of 0.25% Crystal Violet and 20% Ethanol in water to each well in order to stain viable cells.
Let the well-plate sit for 00:05:00 at Room temperature .
Plunge the well-plate into a first bucket filled with 1% bleach diluted in water.
In order to properly clean the plate, plunge the well-plate into a second bucket filled with water.
Count plaques against a white background.