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. 2022 May 1;3(2):101355.
doi: 10.1016/j.xpro.2022.101355. eCollection 2022 Jun 17.

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

Stephanie B Hatch  1 , Remko Prevo  2 , Tiffany Chan  2 , Val Millar  1 , Bart Cornelissen  2 , Geoff Higgins  2 , Daniel Ebner  1
Affiliations

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

Stephanie B Hatch et al. STAR Protoc. .

Abstract

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).

Keywords: Cancer; Cell Biology; Cell culture; Cell-based Assays; High Throughput Screening; Molecular Biology; Single Cell.

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Conflict of interest statement

The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
siRNA library plate layout Controls are located on the edge columns, 1 and 12. A total of eight non-targeting (NT), one PLK1 siRNA (transfection control), three DNA-PKcs siRNA (DN; positive radiosensitization control), and four un-transfected (UN) wells are found in columns 1 and 12. Library siRNA are located in columns 2 through 11.
Figure 2
Figure 2
Mixing map showing quadrants of a single well in a 96-well cell plate
Figure 3
Figure 3
Screenshot of the CHARM settings used for colony counting (image has been enlarged and includes 2 mm scale bar).
Figure 4
Figure 4
Illustration of a representational pair of 96-well 0 Gy and 7 Gy plates
Figure 5
Figure 5
The 22Rv1 prostate carcinoma cell line forms more diffuse colonies than those formed by HeLa cells (the images have been enlarged and include a scale bar in 1000 and 500 um). (A) Example image of Crystal Violet-stained 22Rv1 cells. (B) Image analysis steps.
See this image and copyright information in PMC

References

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    1. Tiwana G.S., Prevo R., Buffa F.M., Yu S., Ebner D.V., Howarth A., Folkes L.K., Budwal B., Chu K.-Y., Durrant L. Identification of vitamin B1 metabolism as a tumor-specific radiosensitizing pathway using a high-throughput colony formation screen. Oncotarget. 2015;6:5978. - PMC - PubMed
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