opera phenix plus high throughput microplate confocal imager  (Revvity)

Journal: NAR Cancer

Article Title: Advancing inducible gene-inactivation systems to explore synthetic lethality

doi: 10.1093/narcan/zcag004

Figure Lengend Snippet: Pipeline of the core single- and co-transfection type IGIS platform. Equal amounts of cells were plated into 6–6 wells per cell line in a 24-well plate. All wells were induced with 1 μg/ml Tet for the rest of the experiment. Medium was changed on the cells on the third day. On the fourth day, half of the samples were treated with a DNA-damaging or a replication stress-inducing agent, while the other half was left untreated. On the second day after treatment, the cells were stained with Hoechst 33342 and propidium iodide. U2OS TetR cells were additionally stained with CellEvent Caspase-3/7 Red detection reagent. After staining, the samples were scanned using a high-throughput scanning microscope. SL: synthetic lethality. Created in BioRender. Kiss, E. (2026) https://BioRender.com/hhc9a6j .

Article Snippet: Samples were scanned with an ImageXpress Micro Confocal high-throughput microscope (Molecular Devices) and analysed with the MetaXpress 6.7.0.211 software package (Molecular Devices).

Techniques: Cotransfection, Staining, High Throughput Screening Assay, Microscopy

Journal: NAR Cancer

Article Title: Advancing inducible gene-inactivation systems to explore synthetic lethality

doi: 10.1093/narcan/zcag004

Figure Lengend Snippet: Pipeline of the single- and co-transfection type f-IGIS platform Equal amounts of cells were plated into four wells of a six-well plate. Next day, cells were (co-)transfected with silencing plasmids. After transfection, all samples were induced with Tet until the end of the experiment. The day after transfection, each well was divided into 6–6 wells of a 24-well plate and put on selective medium (1 μg/ml puromycin + 100 μg/ml hygromycin for U2OS and 300 μg/ml neomycin for RPE1 cells) for 24 h. On the sixth day of the experiment (2 days after the end of selection), half of the samples were treated with 30 μM CP, the other half was left untreated. On the day after treatment (seventh day of the experiment), the cells were stained with Hoechst 33342 and propidium iodide. U2OS TetR cells were additionally stained with CellEvent Caspase-3/7 Red detection reagent. After staining, the samples were scanned with a high-throughput scanning microscope. Only cells showing yellow fluorescent signals were included in the evaluation. SL: synthetic lethality. Created in BioRender. Kiss, E. (2026) https://BioRender.com/714jtg6 .

Article Snippet: Samples were scanned with an ImageXpress Micro Confocal high-throughput microscope (Molecular Devices) and analysed with the MetaXpress 6.7.0.211 software package (Molecular Devices).

Techniques: Cotransfection, Transfection, Selection, Staining, High Throughput Screening Assay, Microscopy

Journal: NAR Cancer

Article Title: Advancing inducible gene-inactivation systems to explore synthetic lethality

doi: 10.1093/narcan/zcag004

Figure Lengend Snippet: Schematic representation of the process of cloning the silencing plasmids. The original pSC1 plasmid contained a neomycin resistance gene (purple box) and an EGFP (light green box), which we switched to a TetO-controlled pH1 promoter (light blue box). Then, we switched the NeoR cassette to puromycin and hygromycin resistance genes (lighter and darker purple boxes, respectively). In the plasmid in which we kept the neomycin resistance, we incorporated the Tet repressor gene (green box). In the next step, we inserted the sh constructs (blue boxes) into all plasmids. To adapt the plasmids to high-throughput microscopy, we inserted YFP-NLS into the NeoR, TRIB-V2 into the HygroR, and MEK1-V1 into the PuroR-containing plasmids (all represented by yellow boxes). Created in BioRender. Kiss, E. (2026) https://BioRender.com/891d8y7 .

Article Snippet: Samples were scanned with an ImageXpress Micro Confocal high-throughput microscope (Molecular Devices) and analysed with the MetaXpress 6.7.0.211 software package (Molecular Devices).

Techniques: Cloning, Plasmid Preparation, Construct, High Throughput Screening Assay, Microscopy