Clonogenic assay
From Wikipedia, the free encyclopedia
Clonogenic assay is a microbiology technique for studying the effectiveness of specific agents on the proliferation of cells. It is frequently employed in cancer research laboratories to determine the effect, if any, of a drug, radiation, or a combination of both on proliferating tumor cells. The term "clonogenic" refers to the fact that these cells are clones of one another.
The experiment involves three major steps:
- The cells to be studied are "plated".
- The treatment is applied.
- The cells are fixed, stained, and counted.
At the conclusion of the experiment, the primary interest is the percentage of cells which survived the treatment. A graphical representation of survival versus drug concentration or dose of ionizing radiation is called a cell survival curve.
Any type of cell could be used in an experiment, but since the goal of these experiments in oncological research is the discovery of more effective cancer treatments, human tumor cells are a typical choice. The cells typically come from prepared "cell lines," which have been well-studied and whose general characteristics are known. The cells are put in petri dishes or in plates which contain several circular "wells." Particular numbers of cells are plated depending on the experiment; for an experiment involving irradiation it is usual to plate larger numbers of cells with increasing dose of radiation. For example, at a dose of 0 or 1 gray of radiation, 500 cells might be plated, but at 4 or 5 grays 2500 might be plated, since very large numbers of cells are killed at this level of radiation and the effects of the specific treatment would be unobservable.
The treatment is usually a drug, ionizing radiation, or both. Some current research studies the potentiation of drug effects by concurrent irradiation -- a synergistic effect -- and in this situation two groups are studied: a control group, which is not treated with the drug; and a treatment group, which is treated with the drug. Both groups are irradiated. If the slopes of their survival curves differ significantly, then a potentiating effect may be evident and could be studied further.
Counting the cell colonies is usually done under a microscope and is quite tedious. Recently, machines have been developed which marry modern optical technology with algorithmic graphical analysis (via software on a connected computer) to automate the counting process. One such machine, Oxford Colcount, works by accepting certain types of cell plates through a slot (not unlike a CD player), taking a special photograph, and uploading it to a computer for immediate analysis. Reliable counts are available in seconds, with a sufficiently fast computer system. At the time of this writing (Spring 2004), these machines are extremely expensive, with basic models retailing for over USD$30,000.
A thorough discussion of the promising research being conducted with the aid of this technique is beyond the scope of this text, but some studies involve the effect of the expression of particular genes or receptors on the cell, the responses of different cell types, or synergistic effects of multiple drugs.
