W587:Kim:A high-throughput colony formation assay for profiling novel compounds and RNAi reagents using the Acumen eX3

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Andrew Goulter, Richard Kim and Jason Mundin

Cell colony formation assays measure a cell's ability to grow unattached to a surface and have applications in a range of areas including hematopoietic stem cell research, cell transformation studies and the prediction of responses of tumors to chemotherapeutic agents.
Traditionally, these assays have been carried out using a semisolid agarose bi-layer system in low density culture plates or petri-dishes with manual enumeration of colonies using a microscope. These methods are both low throughput and based upon subjective determination of the number of colonies thus making them unamenable to high throughput screening.
The objective of this study was to demonstrate that the Acumen platform could quickly and reproducibly quantify the effects of test compounds and RNAi reagents on their ability to affect cell colony formation, and therefore be used as a screening platform.
A549 cells, growing in a agarose gel were incubated for 8 days in the presence of staurosporine, RNAi reagents and respective controls. The assessment included studying the effect of varying initial cell number and output parameters on the quality of the data.
It was shown that colonies seeded at 1,000 cells per well and stained with propidium iodide generated robust data. Using TTP LabTech’s proprietary spherical volume algorithm, the effects of the staurosporine on the colony formation showed a concentration dependent inhibition of cell colony formation; RNAi treatments 2, 3 and 4 caused a large inhibition compared to the control, RNAi 1, all of which were statistically significant from the respective controls.
The results of this study demonstrated that the Acumen eX3 can be used as a high-throughput platform for investigation of effects of test compounds and RNAi reagents on cell colony formation.

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