Soft Agar Assay (Colony Formation Assay)
Cell-Based Assays

Soft Agar Assay (Colony Formation Assay)

The Colony Formation Assay, also called a clonogenic assay, allows screening of the therapeutic efficacy of drugs for anchorage-independent cell growth floating in soft agar. In the Soft Agar Assay, cells grow from single cells to cell colonies in an agar solution keeping them from the solid surface and allows growth in an anchorage-independent way.

The anchorage-independent formation of cell colonies is one of the hallmark characteristics of cellular transformation and uncontrolled cell growth. Normal epithelial cells are supported by basement membranes providing survival and proliferative signals and undergo apoptosis when placed in suspension culture. Tumor cells, in contrast, evade attachment-regulated apoptosis, leading to uncontrolled proliferation. In order to discover agents that revert cell transformation and inhibit anchorage-independent cell growth, the Soft Agar Assay combines sophisticated 3D cell culture with a high throughput setup and reliable quantification.

What is a Soft Agar Assay?

The Soft Agar Assay is one of two 3D assays offered by Reaction Biology. The Soft Agar Assay is used to test the impact of drugs on the proliferation of single cells growing into colonies. In contrast, the 3D Tumor Spheroid Assay is used for testing a drug’s anti-proliferative and cytotoxic capacity on existing tumor spheroids.

Our colony formation assay service provides the following benefits:

  • Longterm exposure: Up to three weeks of compound exposure possible
  • Available for drug combination treatment
  • All cell lines are subject to authentification via STR profiling

Colony Formation

Single cells are seeded in soft agar medium for growth independent of the attachment to a surface. Tumor cells, as opposed to other non-transformed cell types, can divide in an anchorage-independent way with single cells building cell colonies over a course of several days or a few weeks.

H460 cell colonies in the soft agar assay

H460 cell colonies in the soft agar assay

Colony Formation Assay Details

  • Assay Description
  • Example Data
  • Screening Details
Assay Description

soft agar assay procedure for colony formation of tumor cells

Wells of a 96-well plate were coated with 0.6% soft agar (A), followed by seeding of the cells in 0.4% soft agar (B). After the agar has solidified, compounds are added (B) and cells are incubated several days until colonies have formed (C). A cell viability reagent is added, and fluorescence is measured as an indirect quantification of colony growth in soft agar using Resazurin (D).

Example Data

soft agar assay procedure for colony formation of tumor cells

The Aurora B inhibitor VX-680 was tested for inhibition of the soft agar growth of A549 non-small cell lung cancer cells at indicated concentrations. Cells were left to form colonies for 5 days, photographed, and stained with a fluorescent cell viability agent. Fluorescence was quantified and, for analysis of IC50 values, was expressed as a percentage of colony growth in the presence of solvent alone.

Screening Details

Setup: IC50 value determination. Drug combination testing is possible.

Controls: DMSO only is high control. Staurosporine treatment (at 1×10-5M) is low control. In every project, we include the IC50 value determination of a reference compound.

Cell lines: Please inquire to receive a list of cell lines that are established with the Soft Agar Assay format. The establishment of assays with custom cell lines is an option.

Report: A detailed report including assay conditions, methodology, and comprehensive evaluation of data as well as raw data for each analysis will be provided. Photographs of colonies can be provided upon request.

Turnaround time for standard studies is around 3 to 5 weeks. Expedited scheduling and data delivery can be arranged prior to study commencement.

Screening facility: The assay is performed in Freiburg, Germany.

Compound requirements: Compound requirements in brief: 30 µl of the stock solution with 1000x of the highest assay concentration or the equivalent of solid material.