Abstract 5497: An efficient clonogenic assay for cytotoxic drug screening
Authors: Meng Fanying , Bhupathi Deepthi , Hart Charles P. 
Published: February 2011

Abstract
Clonogenic assays are considered to be the “gold standard assay” to test for the effects of drugs and radiation on the growth and proliferative characteristics of cells in vitro. Clonogenic-based assays are considered superior to short-term cell proliferations-based assays as a clonogenic assay is insensitive to p53 status. The clonogenic assay is the measurement of a drug's cytotoxic activity against a single cell population and is the most valid in vitro approach to predict the chemosensitivity of human cancers. The traditional clonogenic assay is very time-consuming and labor-intensive. In addition, the cell disaggregation step necessary for plating in the clonogenic assay results in a loss of cell-cell and cell-substrate interactions which may be critical for the drug response. To overcome the technical challenges and limitations of the traditional clonogenic assay, a 6-well plate clonogenic assay was developed and optimized. Current 6-well clonogenic assays are initiated with a single seeding density, and then surviving cells grow over 10 to 12 days and form colonies. The data generated from this from of the assay only allows the determination of IC50s but not IC90s due to a narrow one log range of cell killing. We have developed an efficient and simple six-well clonogenic assay that yields a wider range of cell killing covering 5 logs. The key is that a series of different cell numbers are seeded in 6 well plates ranging from a density from 100 cells per well to 100,000 cells per well. After a 24 h cell attachment period, cells in each plate were treated with five different concentrations of the test compound. After incubation for the appropriate period of time at 37oC in a 95% air and 5% CO2 atmosphere incubator, drug containing medium was removed and the cells were cultured in fresh medium for additional 10-15 days until adequate colony formation. At the end of incubation, cell colonies were stained with 0.25% w/v crystal violet in 95% Ethanol. After air drying, the plates were scanned with an image-based colony counter (Alpha Innotech) and colonies with more than 50 cells were quantified. To directly compare the experimental results derived from this improved 6-well plate clonogenic assay and the traditional approach to the clonogenic assay, we ran the two forms of the assays simultaneously using cisplatin and SN-38 as the cytotoxic test agents. Cispatin and SN-38 showed comparable IC50, IC90 and IC99 values in both the improved six-well clonogenic assay and the traditional clonogenic assay. In summary, the key advantage of this 6-well clonogenic assay is that it does not involve the trypsinization and replating step after drug treatment and also that is greatly simplifies the traditional clonogenic assay and allows for its use as a moderate-throughput primary drug screening tool.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5497.