Increased efficiency for performing colony formation assays in 96-well plates: novel applications to combination therapies and high-throughput screening

Biotechniques. 2008 Feb;44(2):ix-xiv. doi: 10.2144/000112757.


The colony formation assay (CFA) is the gold standard for measuring the effects of cytotoxic agents on cancer cells in vitro; however, in its traditional 6-well format, it is a time-consuming assay, particularly when evaluating combination therapies. In the interest of increased efficiency, the 6-well CFA was converted to a 96-well format using an automated colony counting algorithm. The 96-well CFA was validated using ionizing radiation therapy on the FaDu (human hypopharyngeal squamous cell) and A549 (human lung) cancer cell lines. Its ability to evaluate combination therapies was investigated by the generation of dose-response curves for the combination of cisplatin and radiation therapy on FaDu and A549 cells. The 96-well CFA was then transferred to a robotic platform for evaluating its potential as a high-throughput screening (HTS) readout. The LOPAC1280 library was screened against FaDu cells, and eight putative hits were identified. Using the 96-well CFA to validate the eight putative chemicals, six of the eight were confirmed, resulting in a positive hit rate of 75%. These data indicate that the 96-well CFA can be adopted as an efficient alternative assay to the 6-well CFA in evaluating single and combination therapies in vitro, providing a possible readout that could be used on a HTS platform.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line, Tumor
  • Cisplatin / pharmacology
  • Combined Modality Therapy
  • Dose-Response Relationship, Drug
  • Dose-Response Relationship, Radiation
  • Humans
  • Reproducibility of Results
  • Small Molecule Libraries / analysis
  • Tumor Stem Cell Assay / instrumentation*
  • Tumor Stem Cell Assay / methods*
  • Tumor Stem Cell Assay / standards


  • Small Molecule Libraries
  • Cisplatin