Aug 03, 2025
Live Virus SARS-CoV-2 - iPSC Pneumocyte - Antiviral Screening
- Briana McGovern1,2
- 1Icahn School of Medicine at Mount Sinai;
- 2ASAP Drug Discovery
- ASAP Discovery
Protocol Citation: Briana McGovern 2025. Live Virus SARS-CoV-2 - iPSC Pneumocyte - Antiviral Screening. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqne6kgk5/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: August 01, 2024
Last Modified: August 03, 2025
Protocol Integer ID: 104490
Keywords: SARS-CoV-2, antiviral, screening assay, cellular, drug discovery, antiviral screening against sar, antiviral screening, antiviral screening the following, live virus sar, antiviral candidate, ipsc pneumocyte cell, ipsc pneumocyte, protocol for live virus, live virus, pneumocyte, matching cytotoxicity, immunofluorescent staining, analysis of immunofluorescent staining, cytotoxicity, drug toxicity, sar, cov
Funders Acknowledgements:
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)
Grant ID: U19AI171399
Disclaimer
The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Abstract
The following is a protocol for live virus antiviral screening against SARS-CoV-2 in iPSC pneumocyte cells. iPSC pneumocytes 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. Each live virus screening assay is accompanied by a matching cytotoxicity screening assay to investigate drug toxicity.
Guidelines
Always wear appropriate PPE for this protocol
Refer to Material Safety Data Sheets for additional safety and handling information.
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
Deep Well Plate (96 well)Thermo FisherCatalog #10045
Formaldehyde
Troubleshooting
Safety warnings
Assay is performed under biosafety level 3 conditions.
Plates
Obtain pneumocyte cells seeded in 96-well plates and appropriate media.
Total amount of plates should be divided in half - one for antiviral screening and one for cytotoxicity screening.
Protocol
CREATED BY
Briana L McGovern
For each plate you can test 3 compounds (in triplicate, 8 dilutions per compound), 2 DMSO controls, and 1 uninfected control in the following layout:
Treatment layout
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 in storage. We recommend organizing the compounds the day before.
These conditions will be different depending on if you are going to perform the serial dilutions manually, or using the Tecan D300e (which is essentially a drug printer).
The protocol will fork into these two methods below.
The serial dilutions will follow different protocols depending on if you will do them by hand or by using a TecanD300e.
Before the assay, you should supplement the provided pneumocyte media with DMSO for your screening. DMSO supplementation will correspond to the highest amount of drug stock used.
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, based on your testing plan, to the first row of the deep well.
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, by hand, from the plates. The pneumocytes are very sensitive so it's best to aspirate the media by hand rather than vacuum line. Work one pair 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
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 concentrations of 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
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.
Prepare your materials for entering the facility. Tips, media, PPE etc.
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
CREATED BY
Briana L McGovern
Infection
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
1d
After 24 hours, add 100ul 1 % (v/v) Formaldehyde 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
CREATED BY
Briana L McGovern
Staining
Follow the SARS-CoV-2 staining protocol
Imaging
Image the infected plates on the
Equipment
Cytation1
NAME
Imager
TYPE
Biotek
BRAND
n/a
SKU
LINK
Analysis