Clonogenic Assay

Introduction
• Clonogenic assay or colony formation assay is an in vitro cell survival assay based on
the ability of a single cell to grow into a colony.
• Clonogenic cell survival is a basic tool that was described in the 1950’s by Puck and
Marcus for the study of radiation effects. Much of the information that has been
generated on the effect of radiation on mammalian cells has been obtained from
clonogenic cell survival assays.
• Clonogenic assays are regarded as the “gold standard” cellular-sensitivity assay.
Principle
• In the standard clonogenic assay, cells in the exponential phase of growth are exposed to
a cytotoxic drug. A cell that is not reproductively dead and has retained the capacity to
divide and proliferate indefinitely can produce a large clone or a large colony of cells
and is then referred to as “clonogenic.” The colony is defined to consist of at least 50
cells.
• The assay essentially tests every cell in the population for its ability to undergo
‘‘unlimited’’ division to determine cell reproductive death after treatment with ionizing
radiation, but can also be used to determine the effectiveness of other cytotoxic agents.
Only a fraction of seeded cells retains the capacity to produce colonies. Before or after
treatment, cells are seeded out in appropriate dilutions to form colonies in 1–3 weeks.
Procedure
• Initial handling of cells
• Clonogenic assay setup
• Fixation and staining of colonies
• Counting of colonies
• Analyzing the results
Clonogenic Assay setup(Methods)
• There are two essentially different ways to perform studies using this assay:
• (A) Plating before treatment
• Cells are plated before treatment. Cells are harvested from a stock culture and plated at
appropriate dilutions into (cluster) dishes. After attachment of the cells to the dishes, which
generally takes 2 h or more, the cells are treated. The treatment has to be performed before
cells start replicating; otherwise, the numbers of cells per dish will increase, yielding more
colonies. After treatment, the dishes are placed in an incubator and left there for a time
equivalent to at least six potential cell divisions. This method is often used for a quick
screening of the sensitivity of cells to different treatments.
Clonogenic Assay setup(Methods)
• (B) Plating after treatment (IP or DP)
• Cells are treated in dishes and subsequently re-plated in appropriate dilutions to assess
clonogenic ability. The replating may be performed immediately after treatment (IP) or it
may be delayed (DP) to allow repair processes. This method is used especially in
radiobiological research to determine potentially lethal- and sublethal damage repair.
Fixation and staining of colonies
• Remove the medium above the cells.
• Rinse carefully with PBS.
• Remove the PBS and add 2–3 ml of a mixture of 6.0% glutaraldehyde and 0.5% crystal violet.
• Leave this for at least 30 min.
• Remove the glutaraldehyde crystal violet mixture carefully and rinse with tap water. Do not
place the dishes or plates under the running tap, but fill the sink with water and immerse the
dishes or plates carefully.
• Leave the dishes or plates with colonies to dry in normal air at room temperature (20 ˚C).
Counting of Colonies
• The standard procedure is to count using a stereomicroscope and an automatic counting
‘‘colony counter pen.’’
• A typical example of the result of a clonogenic assay of SW-1573 human lung tumor
cells is presented.
• Plating efficiency and surviving fraction are determined.
Clonogenic assay performed in six-well plates, with
clones produced by SW-1573 lung tumor cells
Counting of Colonies
• Plating efficiency is the ratio of the number of colonies to the number of cells seeded or
the fraction of colonies from cells not exposed to the treatment.
• Surviving fraction is the number of colonies that arise after treatment of cells, always
calculated taking into account the PE of control cells.
Dose-Response Curve
• A typical dose–response curve obtained by clonogenic assay. The human colon tumor
cell line HT29 in exponential growth was exposed to mitomycin C for 3 h.
Uses
• Clonogenic assays are widely used in the field of cancer research. While this assay was initially
used in the field of radiobiology, it has become a standard tool in cancer research to evaluate
cellular growth and the cytotoxic effects of various agents with potential clinical application.
This includes chemotherapeutic agents and targeted therapies.
• Although colony formation assay is more widely known as a cancer biology assay, it does have
applications in the field of stem cell biology, where the self-renewal potential of stem cells and
their progenitors is assessed.
• Assessing colony formation is most commonly used for hematopoietic stem cells (HSCs), in
the form of a colony formation unit (CFU) assay.
Uses
• Clonogenic growth is used for evaluating the stemness of particular cell populations as
stem cells are long-living cells with the potential for ongoing proliferation.
• This is particularly relevant to cancer research as cancer stem cells are often associated
with chemoresistance, the formation of secondary tumors, and cancer recurrence.
Therefore, investigating cancer-stemness using a clonogenic assay is a valuable and
widely used tool to predict the efficacy of a particular therapy.
References
• Chemosensitivity Volume 1 In Vitro Assays Edited by Rosalyn D. Blumenthal
Garden State Cancer Center, Belleville, NJ
• Cancer Cell Culture Methods and Protocols Edited by Simon P. Langdon
Western General Hospital Edinburgh, UK
• https://www.researchgate.net/publication/6416427_Clonogenic_assay_of_cells
• Clonogenic assay: what, why and how By Jenna Bleloch, Ph.D.