Automatic cell cloning assay for determining the clonogenic capacity of cancer and cancer stem-like cells

Authors

  • Radek Fedr,

    1. Department of Cytokinetics, Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., Brno, Czech Republic
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  • Zuzana Pernicová,

    1. Department of Cytokinetics, Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., Brno, Czech Republic
    2. Center of Biomolecular and Cellular Engineering, International Clinical Research Center, St. Anne's University Hospital Brno, Brno, Czech Republic
    3. Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic
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  • Eva Slabáková,

    1. Department of Cytokinetics, Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., Brno, Czech Republic
    2. Center of Biomolecular and Cellular Engineering, International Clinical Research Center, St. Anne's University Hospital Brno, Brno, Czech Republic
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  • Nicol Straková,

    1. Department of Cytokinetics, Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., Brno, Czech Republic
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  • Jan Bouchal,

    1. Department of Clinical and Molecular Pathology, Faculty of Medicine and Dentistry, Palacky University Olomouc, Czech Republic
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  • Michal Grepl,

    1. Department of Urology, Faculty of Medicine and Dentistry, Palacky University Olomouc, Czech Republic
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  • Alois Kozubík,

    1. Department of Cytokinetics, Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., Brno, Czech Republic
    2. Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic
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  • Karel Souček

    Corresponding author
    1. Department of Cytokinetics, Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., Brno, Czech Republic
    2. Center of Biomolecular and Cellular Engineering, International Clinical Research Center, St. Anne's University Hospital Brno, Brno, Czech Republic
    • Ph.D., Institute of Biophysics, Academy of Sciences of the Czech Republic, Královopolská 135, CZ-612 65 Brno, Czech Republic
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Abstract

The clonogenic assay is a well-established in vitro method for testing the survival and proliferative capability of cells. It can be used to determine the cytotoxic effects of various treatments including chemotherapeutics and ionizing radiation. However, this approach can also characterize cells with different phenotypes and biological properties, such as stem cells or cancer stem cells. In this study, we implemented a faster and more precise method for assessing the cloning efficiency of cancer stem-like cells that were characterized and separated using a high-speed cell sorter. Cell plating onto a microplate using an automatic cell deposition unit was performed in a single-cell or dilution rank mode by the fluorescence-activated cell sorting method. We tested the new automatic cell-cloning assay (ACCA) on selected cancer cell lines and compared it with the manual approach. The obtained results were also compared with the results of the limiting dilution assay for different cell lines. We applied the ACCA to analyze the cloning capacity of different subpopulations of prostate and colon cancer cells based on the expression of the characteristic markers of stem (CD44 and CD133) and cancer stem cells (TROP-2, CD49f, and CD44). Our results revealed that the novel ACCA is a straightforward approach for determining the clonogenic capacity of cancer stem-like cells identified in both cell lines and patient samples. © 2013 International Society for Advancement of Cytometry

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