Feb 11, 2026

Public workspaceVarChamp Cell Painting

DOI
https://dx.doi.org/10.17504/protocols.io.36wgqnwkxgk5/v1
  • Tanisha Teelucksingh1,
  • Florent Laval2
  • 1University of Toronto;
  • 2Center for Cancer Systems Biology (CCSB), Dana-Farber Cancer Institute, Boston, MA
Tanisha Teelucksingh
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DOI: https://dx.doi.org/10.17504/protocols.io.36wgqnwkxgk5/v1
Protocol Citation: Tanisha Teelucksingh, Florent Laval 2026. VarChamp Cell Painting . protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqnwkxgk5/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: In development
We are still developing and optimizing this protocol
Created: October 08, 2024
Last Modified: February 11, 2026
Protocol Integer ID: 109318
Keywords: varchamp cell painting, missense variants on protein, subcellular localization, missense variant
Funders Acknowledgements:
National Human Genome Research Institute (NHGRI)
Grant ID: UM1HG011989
Abstract
Assessment of the impact of missense variants on protein subcellular localization
Troubleshooting
Virus Production - Seeding transfection plates (96-well format)
Seed HEK293T Cells - 96-well format,Amount100 µL per well. Media used: DMEM
Note
All media contains 10% FBS and 5% penicillin streptomycin

  1. if transfecting packaging plasmids on the same day, seed 2.4x104 cells/well. Allow at least Duration04:00:00 for cells to adhere, incubating at Temperature37 °C , 5% CO2
  2. if transfecting packaging plasmids the next day, seed 1.2x104 cells/well. Incubate DurationOvernight at Temperature37 °C , 5% CO2
4h 30m
Remember to harvest mycoplasma sample for testing downstream
Critical
Virus Production - Preparing packaging/transfection mix (96-well format)
Prepare packaging mix - Amount10 µL /well
1. 50ng/well psPAX2
2. 5ng/well pVSV-G
3. Top up to Amount10 µL /well with OptiMEM
Prepare transfection mix - Amount10 µL /well
1.Amount9.4 µL OptiMEM/well
2. Amount0.6 µL TransIT/well
Note
Make sure to add the TransIT drop wise and mix well

Critical
Prepare 96-well plate - 33.33 uL total volume per well
1. 10uL packaging mix
2. 10uL transfection mix
3. 10uL sterile water
4. 3.33uL lentiviral expression plasmid (normalized to 15ng/uL)
5. Incubate at room temperature forDuration00:30:00
Note
Mix well before incubation

30m
Incubation
Temperature
Transfer entire mix (33.33uL) to HEK293T cells
1. Incubate for Duration48:00:00 at 37ºC, 5% CO2

2d
Transduction of U2OS cells
Seed U2OS Cells - 384-well format, 40uL per well. Media used: McCoy's 5A with 10% FBS and 1% penicillin streptomycin
1. Seed 1000 cells/well --> include 10ug/mL polybrene
2. Allow cells to settle for ~ 1 hour at room temperature before incubating at 37ºC, 5% CO2 for an additional 3 hours
Remember to harvest mycoplasma sample for testing downstream
Critical
Spinfection - 384-well format
1. Add 6uL of virus per well
2. Spin plates at Centrifigation1200 x g, 37°C for 30 minutes
3. Incubate for 48 hours at 37ºC, 5% CO2
Centrifigation
Temperature
Puromycin Selection of Transduced U2OS cells
Dilute puromycin in media (McCoy's 5A with 10% FBS and 1% penicillin streptomycin) to a final concentration of 1.5ug/mL
1. Flick media from 384-well plates
2. Add 30uL of diluted puromycin to each well
3. Incubate for Duration48:00:00 at 37ºC, 5% CO2
4. Make sure to monitor untransduced control for complete selection
2d
Incubation
Overnight
Mycoplasma Testing
Before proceeding with staining and fixation of cells, samples taken on steps 2 and 8 should be tested for mycoplasma as this will impact downstream applications
Critical
Mitotracker Staining
Dilute MitoTracker Deep Red in McCoy's 5A ( with 10% FBS and 1% penicillin streptomycin) to a final concentration of 500nM
1. Flick media from 384-well plates
2. Add 20uL of diluted MitoTracker Deep Red to each well
3. Incubate for 1 hour at 37ºC, 5% CO2
Cell Fixation
Dilute 32% paraformaldahye to a final concentration of 4% in McCoy's 5A ( with 10% FBS and 1% penicillin streptomycin)
1. Flick media from 384-well plates
2. Add 20uL of diluted PFA to each well
3. Cover with tin foil - PFA is light sensitive
4. Incubate for 15-30 minutes at room temperature
5. Wash each plate 4 times with 1x HBSS
6. If not staining right away, store plates with 50uL 1x HBSS per well, covered in tin foil at 4ºC
Pause
Cell Staining
Prepare Staining and Permeabilization Buffer in 1x HBSS:
1. 0.1% Triton (vol/vol)
2. 1% BSA (wt/vol)
Dilute dyes to the following concentrations:
1. Phalloidin 568 - 0.125uL/mL (stock 6.6uM)
2. Hoechst 33342 - 1ug/mL (stock 10mg/mL)
3. WGA 555 - 1.5ug/mL (stock 1mg/mL)
Note
All dyes are spun down before use according to Cell Painting Protocol V3

Flick out 1x HBSS from cells after fixation
1. Add 20uL of staining solution per well
2. Centrifuge at 500 x g for 1 min to remove air bubbles
3. Incubate, covered in tin foil for 30 minutes
4. Wash each plate 4 times with 1x HBSS
5. Leave 50uL 1x HBSS per well
Imaging
All confocal images are captured on a Perkin Elmer Opera Phenix Microscope with the following conditions:
1. 20X water objective
2. Single plane (-7um)
3. 384 wells, 9 fields
4. Separate all channels:
ChannelExcitationEmissionExposurePower
DAPI405435-480100 ms60%
Alexa488488500-55060 ms30%
Alexa568561571-59660 ms30%
Mitotracker Deep Red640650-76020 ms20%
Brightfield HighTransmission100 ms50%
Brightfield Transmission100 ms50%
Brightfield LowTransmission100 ms50%

Note
Current run time is 2hr 12min for 1 plate