Colony Formation Assays

The colony formation assay, or clonogenic assay, evaluates the proliferative capacity of a single cell. For applications such as cancer drug screening, it is important to distinguish cells that retain this proliferative capacity from those that do not. Conventional analysis of this assay involves scoring and quantifying colonies in each well of a multi-well format manually by eye, limiting its throughput capabilities and reproducibility.

Schematic representation of the colony formation assay.

Figure 1. Schematic representation of the colony formation assay. Cells in suspension are seeded in tissue culture wells at a low enough density to enable single cells to proliferate into clonal populations. Colonies are defined as consisting of a defined number of cells (e.g. >50 cells). The potency of anti-proliferative compounds can be assessed based on the number of surviving colonies relative to control.


Automated colony formation assay using fluorescence imaging and Gen5 analysis

A robust and convenient method for conducting and analyzing the colony formation assay in a range of microplate formats, from 6- to 96-well microplate densities, using fluorescent cell labels and automated fluorescence microscopy. Accurate and objective determination of colony number is achieved using direct cell counts and subpopulation analysis. 

Automated fluorescent colony identification and characterization using Crystal Violet and Hoechst 33342 staining

Figure 2. Automated fluorescent colony identification and characterization using Crystal Violet and Hoechst 33342 staining. A’-E’ are insets of the indicated region in A-E, respectively. (A) Whole wells of a 96-well microplate are captured with a 2x2 montage that undergoes a background reduction step and stitching. (B) Colony Identification is possible using the fluorescent signal of Crystal Violet as the primary mask in the Cellular Analysis data reduction step. (C) Nuclei are visualized with Hoechst 33342 staining, (D) and are identified as a secondary mask within each colony using the Spot Counting module, allowing for # nuclei/colony to be quantified. (E) A sub-population analysis is set to apply a cut-off for colony size based on # of nuclei. The number of nuclei detected are indicated. Purple = colonies with >32 nuclei/cells (>5 rounds of division).


Using the Colony Formation Assay to Establish IC50 Values for Anti-Cancer Compounds

IC50 determination of doxorubicin in a 6-well and 96-well formats.

Figure 3. IC50 determination of doxorubicin in a 6-well and 96-well formats. The colony formation assay was conducted where Caco2 and HeLa cells were seeded in either 6-well plates or (A) 96-well microplates and cultured for 7 days in the presence of increasing concentrations of known antiproliferative compounds. Automated upright color brightfield microscopy and cellular analysis of crystal violet-stained colonies was performed using the Cytation 7 Multi-Mode reader with Wide Field of View. Dose response curves and calculated IC50 values were compared across drug treatments and microplate formats.

Increase assay throughput and performance

BioSpa™ Compatible Assay: Live-cell analysis on up to 8 microplates
The BioSpa Live Cell Analysis System
supports multi-plate colony formation assay applications.



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For Research Use Only. Not for use in diagnostic procedures.