Mar 25, 2025

Public workspaceVariant painting via fluorescence

DOI
https://dx.doi.org/10.17504/protocols.io.5jyl8erydl2w/v1
  • Chloe Reno1
  • 1University of Toronto
Chloe Reno
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DOI: https://dx.doi.org/10.17504/protocols.io.5jyl8erydl2w/v1
Protocol Citation: Chloe Reno 2025. Variant painting via fluorescence. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl8erydl2w/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: March 25, 2025
Last Modified: March 25, 2025
Protocol Integer ID: 124928
Keywords: variant painting via immunofluorescence, cell painting protocol v3, variant painting, variation on protein localization, immunofluorescence, protein localization, protein, imaging, fluorescence, throughput imaging platform, cell, coding variation, additional cellular compartment, mitochondria
Funders Acknowledgements:
NIH IGVF
Grant ID: HG011989
Abstract
High-throughput imaging platform to assay the impact of coding variation on protein localization. Proteins are fused to mNeonGreen for visualization, alongside additional cellular compartments including the nucleus, mitochondria, actin, golgi, and plasma membrane. This protocol is adapted from Variant Painting via immunofluorescence and Cell Painting Protocol v3.

Troubleshooting
Day 1: Cell Seeding
Seed 1.2x104 HEK293T cells/well in 96-well plate, Amount100 µL per well. Media is high glucose DMEM supplemented with 10% FBS, 1% pepenicillin-streptomycin. Incubate DurationOvernight at Temperature37 °C , 5% CO2.
Note
Harvest a sample of HEK293T cells for mycoplasma testing.

Note
If performing same-day transfection, double the seeding density and allow cells to adhere for ~4 hrs.


Day 2: Viral Packaging
2d
Prepare packaging master mix:
1. Amount50 ng /well psPAX2
2. Amount5 ng /well pVSV-G
3. Top up to Amount10 µL /well with OptiMEM

Prepare transfection reagent master mix:
1. Amount9.4 µL /well OptiMEM
2. Amount0.6 µL /well TransIT-LT1 Transfection Reagent

Prepare 96-well transfection complexes:
1. Add Amount10 µL /well molecular grade water
2. Add Amount3.33 µL /well lentiviral expression plasmid (normalized to 15 ng/uL)
3. Add Amount10 µL /well packaging master mix
4. Add Amount10 µL /well reagent master mix
Incubate at room temperature Duration00:30:00 .

Transfer transfection complexes to seeded HEK293T cells. Incubate Duration48:00:00 at Temperature37 °C , 5% CO2.
Note
Make sure to monitor EGFP transfection control for transfection efficiency.


2d
Day 4: Transduction
2d 4h 30m
Seed 1000 U2OS cells/well, Amount40 µL /well in 384-well black optically clear flat-bottom PhenoPlates. McCoy's 5A m Test before edia is supplemented with 10 ug/mL polybrene + 10% FBS + 1% penicillin-streptomycin.
Allow cells to adhere for Duration01:00:00 at room temperature before incubating at Temperature37 °C , 5% CO2 for Duration03:00:00 .
Note
Harvest a sample of U2OS cells for mycoplasma testing. Test before Day 8.


4h
Perform Spinfection:
1. Add Amount6 µL virus /well
2. Spin plates at Centrifigation1200 x g, 37°C, 00:30:00
3. Incubate Duration48:00:00 at Temperature37 °C , 5% CO2.

2d 0h 30m
Day 6: Puromycin Selection
2d
Prepare 1.5 ug/mL puromycin in McCoy's 5A media supplemented with 10% FBS + 1% penicillin-streptomycin.
1. Flick media out of 384-well imaging plates into a bleach bucket.
2. Add Amount30 µL puromycin media /well
3. Incubate Duration48:00:00 at Temperature37 °C , 5% CO2.
Note
Make sure to monitor untransduced (EGFP transfection) control for complete selection


2d
Day 8: Fixation & Staining
2h 16m
Stain live cells with MitoTracker DeepRed:
1. Dilute MitoTracker DeepRed to Concentration500 nanomolar (nM) in McCoy's 5A + 10% FBS + 1% penicillin-streptomycin.
2. Flick off media from imaging plates.
3. Add Amount20 µL /well of prepared MitoTracker media.
4. Incubate Duration01:00:00 at Temperature37 °C , 5% CO2.

1h
Fix cells:
1. Dilute 32% Paraformaldehyde to 4% in McCoy's 5A + 10% FBS + 1% penicillin-streptomycin.
2. Flick off media from imaging plates.
3. Add Amount20 µL /well of diluted PFA.
4. Wrap plates in foil to protect from light.
5. Incubate Duration00:15:00 - Duration00:30:00 at room temperature.
6. Wash with plate washer 4x with 1xHBSS, including final aspiration.
Note
If you are not staining & imaging the same day, do not include the final apsiration. Instead leave ~Amount50 µL of HBSS behind and store plates at Temperature4 °C wrapped in foil.



45m
Prepare your blocking & permeabilization buffer:
0.1%Triton-X(vol/vol) + 1% BSA (wt/vol) in 1xHBSS.
Prepare your staining solution. Dilute the following dyes in your blocking & permeabilization buffer:
1. Spin down all dyes
2. Dilute Phalloidin AlexaFluor 568 to 8.25 nM
3. Dilute Hoechst 33342 to 1 ug/mL
4. Dilute WGA AlexaFluor 555 to 1.5 ug/mL
Stain cells:
1. Flick off media from imaging plates.
2. Add Amount20 µL /well of staining solution.
3. CentrifugeCentrifigation500 x g, 00:01:00 to remove bubbles.
5. Wrap plates in foil to protect from light. Incubate Duration00:30:00 at room temperature.
6. Wash with plate washer 4x with 1xHBSS, excluding final aspiration t leave ~Amount50 µL /well.

31m
Image:
Confocal images are captured on a PerkinElmer Opera Phenix microscope with the following conditions:
1. 20x water objective
2. Single plane
3. 384-wells, 9 fields
4. Separate Channels:
ChannelExcitationEmissionExposurePower
DNA405435-480100 ms60%
Protein488500-55060 ms30%
AGP561571-59660 ms30%
Mitochondria64076020 ms20%