Automated 96-well format high throughput colony formation assay for siRNA library screen

The colony formation assay is the gold-standard technique to assess cell viability after treatment with cytotoxic reagents, ionizing radiation, and cytotoxic combinatorial treatments. This protocol describes a high-throughput automated and high-content imaging approach to screen siRNA molecular libr...

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Bibliographic Details
Published in:STAR protocols Vol. 3; no. 2; p. 101355
Main Authors: Hatch, Stephanie B., Prevo, Remko, Chan, Tiffany, Millar, Val, Cornelissen, Bart, Higgins, Geoff, Ebner, Daniel
Format: Journal Article
Language:English
Published: United States Elsevier Inc 17.06.2022
Elsevier
Subjects:
Cancer
Cell Biology
Cell culture
Cell-based Assays
Gene Library
High Throughput Screening
High-Throughput Screening Assays - methods
Molecular Biology
Protocol
Radiation, Ionizing
RNA, Small Interfering - genetics
Single Cell
Transfection
Cell culture
Molecular Biology
Cell-based Assays
High Throughput Screening
Single Cell
Cell Biology
Cancer
ISSN:2666-1667, 2666-1667
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Summary:The colony formation assay is the gold-standard technique to assess cell viability after treatment with cytotoxic reagents, ionizing radiation, and cytotoxic combinatorial treatments. This protocol describes a high-throughput automated and high-content imaging approach to screen siRNA molecular libraries in HeLa cervical cancer cells in 96-well format. We detail reverse transfection of cells with siRNAs, followed by ionizing radiation, fixing, and staining of the plates for automated colony counting. This protocol can be used across a broad range of cell types. For complete details on the use and execution of this protocol, please refer to Tiwana et al. (2015). [Display omitted] •High throughput screening (HTS) colony formation assay using liquid handling robots•Screens siRNA molecular libraries in HeLa cervical cancer cells in 96-well format•Automated colony counting and high content imaging readout approaches•Statistical analysis for HTS quality control and quantification Publisher's note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. The colony formation assay is the gold-standard technique to assess cell viability after treatment with cytotoxic reagents, ionizing radiation, and cytotoxic combinatorial treatments. This protocol describes a high-throughput automated and high-content imaging approach to screen siRNA molecular libraries in HeLa cervical cancer cells in 96-well format. We detail reverse transfection of cells with siRNAs, followed by ionizing radiation, fixing, and staining of the plates for automated colony counting. This protocol can be used across a broad range of cell types.
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ISSN:2666-1667
2666-1667
DOI:10.1016/j.xpro.2022.101355