Mar 18, 2025
Live Virus SARS-CoV-2 - HeLa-ACE2 - Antiviral Screening Assay
- 1Icahn School of Medicine at Mount Sinai;
- 2ASAP Discovery Consortium
Protocol Citation: Briana L McGovern 2025. Live Virus SARS-CoV-2 - HeLa-ACE2 - Antiviral Screening Assay. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvmee4ng3p/v1
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: June 05, 2024
Last Modified: March 18, 2025
Protocol Integer ID: 101260
Keywords: antiviral, live virus, screening, drug discovery, cytotoxicity
Funders Acknowledgements:
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)
Grant ID: U19AI171399
Disclaimer
Assay is performed under biosafety level 3 conditions.
Abstract
The following is a protocol for live virus antiviral screening against SARS-CoV-2 in vitro. Cells are prophylactically treated with antiviral candidates that have been serially diluted to investigate activity, via IC50 values generated via analysis of immunofluorescent staining, against SARS-CoV-2.
You may opt to include PgP inhibitor in the assay by adding 2uM into the infection media.
Each live virus screening assay is accompanied by a matching cytotoxicity screening assay in uninfected cells to investigate drug toxicity.
Materials
10% Media
2% Media
96-well plates
96-well deep well plates
12-channel 200/300ul multichannel pipette
200/300ul filtered tips
Compounds of interest
Virus(es) of interest
PPE
reservoir
Protocol materials
Formaldehyde
Deep Well Plate (96 well)Thermo FisherCatalog #10045
Deep Well Plate (96 well)Thermo FisherCatalog #10045
Seeding
Seeding
The day before your assay, seed 96-well plates with HeLa-ACE2 cells at 4,000 cells/well using standard 10% Growth Media.
You will need to make one pair of plates for every 3 compounds. One plate is the antiviral plate that will be infected, and the other is the cytoxicity plate that will measure the toxicity of the antiviral candidates.
For each pair you can test 3 compounds (in triplicate, 8 dilutions per compound), 2 DMSO controls, and 1 uninfected control in the following layout:
Treatment layout
Protocol
NAME
Cytotoxicity Screening Assay - Paired with Antiviral Assays
CREATED BY
Briana L McGovern
Setting up the conditions
Setting up the conditions
Create a screening priority list ahead of time. This list will have the compounds of interest, what maximum concentration you'd like to screen at, and where the compounds are located. We recommend organizing the compounds the day before.
The serial dilutions will follow different protocols depending on if you will do them by hand or by using a
Equipment
Tecan D300e
NAME
Liquid Handler
TYPE
Tecan
BRAND
30100152
SKU
https://lifesciences.tecan.com/products/liquid_handling_and_automation/tecan_d300e_digital_dispenser
LINK
Before the assay, you should prepare enough 2% Media supplemented with DMSO for your screening. DMSO supplementation will correspond to the highest amount of drug stock used.
Include 2uM PgP inhibitor in this infection media if you'd like.
Manual21 steps
Performing the serial dilutions manually.
If your lab does not have a Tecan D300e machine you will need to do the dilutions by hand.
Remove your compounds from -20 to thaw while you set up.
Prepare the Deep Well Plate (96 well)Thermo FisherCatalog #10045 according to this diagram:
Deep well set up for manual serial dilutions
The first row will be the [max] of your compounds. This row will contain 1100ul of 2% media without DMSO. The media for this row does not receive any DMSO since you will add drug dissolved in DMSO directly to it.
The remaining rows of the plate will be filled with 750ul 2% media + supplemented with DMSO.
These volumes are enough to treat both the antiviral and cytotoxicity plates.
Perform the serial dilutions:
Add the compounds of interest to the first row of the deep well (containing 1100ul media).
mix well and move 375ul from row 1 to the row 2. This is your first 1:3 dilution.
Schematic of the manual serial dilutions
Repeat until the entire deep well has been done.
You now have 8 concentrations of your compounds of interest.
Remove the growth media from the plates. Work one pair of plates at a time to prevent drying of the cells.
Add 100ul of the serial dilutions to the wells in the following set up: You will perform this for both the infection plate and the matching cytotoxicity plate
Treatment layout of the HeLa-ACE2 plates.
Notice how the inner columns of the plate receive experimental treatment, while the outer columns are the controls.
You should always have the plates in the correct orientation (top left corner should be A1) so that you are sure where the highest and lowest [compound] are.
Incubate the cells at 37 °C 5% CO2 for 02:00:00 .
This pretreatment time frame could differ depending on what sort of compounds you'd like to test.
2h
Prepping for infection
Prepping for infection
1h 30m
1h 30m
After approximately 01:30:00 you should begin prepping to head up to the BSL-3 facility, where you will infect the plates with SARS-CoV-2.
1h 30m
Calculate the dilution of the virus(es) of interest and bring the calculations with you.
Protocol
NAME
Calculating Multiplicity of Infection (MOI)
CREATED BY
Briana L McGovern
Prepare your materials for entering the facility.
Before heading up to infect the plates, you should perform the mock infection on the cytoxicity plates. We want both plates to match so that we can compare them later.
Add 50ul 2% media to the wells.
Protocol
NAME
Cytotoxicity Screening Assay - Paired with Antiviral Assays
CREATED BY
Briana L McGovern
Infection
Infection
1d
1d
Go to the BSL-3 facility. Perform the entering procedures.
Retrieve your virus(es) from the -80.
Thaw your virus(es) and create your dilution(s)
Using the reservoir, infect your plates (except the uninfected control column) with 50ul of the virus dilution.
Incubate at 37 °C for 24:00:00
1d
Fixing
Fixing
1d
1d
After 24 hours, add 100ul 1 % (v/v) FormaldehydeContributed by users to all wells on the plate.
Spray each lid gently with ethanol and replace.
Double bag the plates in red biohazard bags to bring them out of the facility.
Incubate for 24:00:00 at room temperature inside the bags
1d
In parallel, process the cytotoxicity plates.
Protocol
NAME
Cytotoxicity Screening Assay - Paired with Antiviral Assays
CREATED BY
Briana L McGovern
Staining
Staining
Follow the SARS-CoV-2 Antiviral Staining Protocol
Protocol
NAME
SARS-CoV-2 Antiviral Immunofluorescence staining Protocol
CREATED BY
Briana L McGovern
Imaging
Imaging
Image the infected plates on a plate reader. We use a Cytation1
Equipment
Cytation1
NAME
Imager
TYPE
Biotek
BRAND
n/a
SKU
LINK
Analysis
Analysis
Protocol
NAME
Generating Antiviral & Cytotoxicity Curves
CREATED BY
Briana L McGovern