Mar 25, 2025
Variant painting via fluorescence
- 1University of Toronto
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 24, 2025
Last Modified: March 25, 2025
Protocol Integer ID: 124928
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.
Day 1: Cell Seeding
Day 1: Cell Seeding
Seed 1.2x104 HEK293T cells/well in 96-well plate, 100 µL per well. Media is high glucose DMEM supplemented with 10% FBS, 1% pepenicillin-streptomycin. Incubate Overnight at 37 °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
Day 2: Viral Packaging
2d
2d
Prepare packaging master mix:
1. 50 ng /well psPAX2
2. 5 ng /well pVSV-G
3. Top up to 10 µL /well with OptiMEM
Prepare transfection reagent master mix:
1. 9.4 µL /well OptiMEM
2. 0.6 µL /well TransIT-LT1 Transfection Reagent
Prepare 96-well transfection complexes:
1. Add 10 µL /well molecular grade water
2. Add 3.33 µL /well lentiviral expression plasmid (normalized to 15 ng/uL)
3. Add 10 µL /well packaging master mix
4. Add 10 µL /well reagent master mix
Incubate at room temperature 00:30:00 .
Transfer transfection complexes to seeded HEK293T cells. Incubate 48:00:00 at 37 °C , 5% CO2.
Note
Make sure to monitor EGFP transfection control for transfection efficiency.
2d
Day 4: Transduction
Day 4: Transduction
2d 4h 30m
2d 4h 30m
Seed 1000 U2OS cells/well, 40 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 01:00:00 at room temperature before incubating at 37 °C , 5% CO2 for 03:00:00 .
Note
Harvest a sample of U2OS cells for mycoplasma testing. Test before Day 8.
4h
Perform Spinfection:
1. Add 6 µL virus /well
2. Spin plates at 1200 x g, 37°C, 00:30:00
3. Incubate 48:00:00 at 37 °C , 5% CO2.
2d 0h 30m
Day 6: Puromycin Selection
Day 6: Puromycin Selection
2d
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 30 µL puromycin media /well
3. Incubate 48:00:00 at 37 °C , 5% CO2.
Note
Make sure to monitor untransduced (EGFP transfection) control for complete selection
2d
Day 8: Fixation & Staining
Day 8: Fixation & Staining
2h 16m
2h 16m
Stain live cells with MitoTracker DeepRed:
1. Dilute MitoTracker DeepRed to 500 nanomolar (nM) in McCoy's 5A + 10% FBS + 1% penicillin-streptomycin.
2. Flick off media from imaging plates.
3. Add 20 µL /well of prepared MitoTracker media.
4. Incubate 01:00:00 at 37 °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 20 µL /well of diluted PFA.
4. Wrap plates in foil to protect from light.
5. Incubate 00:15:00 - 00:30:00 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 ~50 µL of HBSS behind and store plates at 4 °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 20 µL /well of staining solution.
3. Centrifuge500 x g, 00:01:00 to remove bubbles.
5. Wrap plates in foil to protect from light. Incubate 00:30:00 at room temperature.
6. Wash with plate washer 4x with 1xHBSS, excluding final aspiration t leave ~50 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:
| Channel | Excitation | Emission | Exposure | Power | |
| DNA | 405 | 435-480 | 100 ms | 60% | |
| Protein | 488 | 500-550 | 60 ms | 30% | |
| AGP | 561 | 571-596 | 60 ms | 30% | |
| Mitochondria | 640 | 760 | 20 ms | 20% |