Aug 03, 2025
iPSC Pneumocyte Live Virus SARS-CoV-2 Cytotoxicity Screening Assay
- Briana McGovern1,2,
- Kris White1,2
- 1Icahn School of Medicine at Mount Sinai;
- 2ASAP Discovery Consortium
- ASAP Discovery
Protocol Citation: Briana McGovern, Kris White 2025. iPSC Pneumocyte Live Virus SARS-CoV-2 Cytotoxicity Screening Assay. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5rm96g1b/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 08, 2024
Last Modified: August 03, 2025
Protocol Integer ID: 104929
Keywords: SARS-CoV-2, antiviral, cytotoxicity, cellular, ipsc pneumocyte live virus sar, based antiviral assay, antiviral assay, antiviral activity, antiviral activity of compound, live virus sar, antiviral, relevant viral antigen, response with concurrent cytotoxicity assay, concurrent cytotoxicity assay, infected cell, pneumocyte, influenza virus, uninfected cell, identification of infected cell, uninfected cell control, live virus, infection control, screening assay, cytotoxicity, immunofluorescence, gp inhibitor, infection, ipsc pneumocyte
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
These immunofluorescence-based antiviral assays rely on identification of infected cells through immunostaining the NP (influenza virus), N (SARS-CoV-2) proteins, or any relevant viral antigen at 488nM and counterstaining with DAPI. Infected cells (488nM) and total cells (DAPI) are identified and
quantified by a plate cytometer (Biotek Cytation 1). Compounds are tested in a triplicate 8-point
dose-response with concurrent cytotoxicity assays (MTT Roche/MTS Promega) in uninfected
cells. These assays can be run on any adherent cell type, such as standard cell lines and iPSC-derived cell types. DMSO infection controls (negative) and uninfected cell controls (positive) should be included on every plate. The DMSO controls will establish 100% infection and the uninfected wells 0% infection for each plate. Relevant control compounds should be included as controls for every run. The effect p-gp inhibitors on known p-gp subtrates should be explored in all new cell types used.
These assays accurately reflect the antiviral activity of compounds in medium-to-high throughput, depending on the level of automation.
Protocol materials
Deep Well Plate (96 well)Thermo FisherCatalog #10045
Troubleshooting
Safety warnings
Always wear appropriate PPE for this protocol
Refer to Material Safety Data Sheets for additional safety and handling information.
Plates
This assay is typically run in parallel to the antiviral screening protocol, so you will end up having 1 pair of plates for every 3 compounds that you want to test. The pair will be identical; one will be infected to test antiviral activity, while the other will remain uninfected to test cytotoxicity.
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 antiviral screening.
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. 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 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.
ManualFrom 13 to 15 steps
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, 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
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, but 2 hours is standard.
2h
Mock Infection
While these cytotoxicity plates will not be infected with virus, they need to be mock infected to match the antiviral plates.
Add 50ul 2% media to the wells.
24 hours post infection, you will fix both the antiviral and cytotoxicity plates. The method you use for the cytotoxicity plates will depend on which reagents you have.
Roche MTT Kit6 steps
Sigma #11465007001
add 10ul MTT reagent.
Incubate 3-4 hours at 37C
Add 100ul SDS reagent and incubate at RT ON
Reading the plates
Read the plates using a plate reader. We use the Cytation1.
read at 560nm
read again at 750nm
Equipment
Cytation1
NAME
Imager
TYPE
Biotek
BRAND
n/a
SKU
LINK
Export the data to excel
Analysis