1. Automatic cell cloning assay for determining the clonogenic capacity of cancer and cancer stem-like cells
- Author
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Radek Fedr, Alois Kozubík, Michal Grepl, Karel Souček, Zuzana Pernicová, Nicol Straková, Jan Bouchal, and Eva Slabáková
- Subjects
Male ,Histology ,Cell Survival ,In Vitro Techniques ,Integrin alpha6 ,Pathology and Forensic Medicine ,03 medical and health sciences ,0302 clinical medicine ,Cancer stem cell ,Antigens, CD ,Antigens, Neoplasm ,Cell Line, Tumor ,medicine ,Biomarkers, Tumor ,Cytotoxic T cell ,Humans ,AC133 Antigen ,Clonogenic assay ,Tumor Stem Cell Assay ,030304 developmental biology ,Cell Proliferation ,Glycoproteins ,0303 health sciences ,biology ,CD44 ,Cancer ,Prostatic Neoplasms ,Cell Biology ,Cell sorting ,medicine.disease ,Flow Cytometry ,Molecular biology ,3. Good health ,Hyaluronan Receptors ,030220 oncology & carcinogenesis ,Colonic Neoplasms ,biology.protein ,Cancer research ,Neoplastic Stem Cells ,Stem cell ,Peptides ,Cell Adhesion Molecules - 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.
- Published
- 2012