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.

• MeSH
• AC133 Antigen MeSH
• Hyaluronan Receptors metabolism   MeSH
• Antigens, Neoplasm metabolism   MeSH
• Antigens, CD metabolism   MeSH
• Glycoproteins metabolism   MeSH
• Integrin alpha6 metabolism   MeSH
• Humans MeSH
• Cell Adhesion Molecules metabolism   MeSH
• Biomarkers, Tumor metabolism   MeSH
• Cell Line, Tumor MeSH
• Neoplastic Stem Cells metabolism   pathology   MeSH
• Prostatic Neoplasms metabolism   pathology   MeSH
• Colonic Neoplasms metabolism   pathology   MeSH
• Peptides metabolism   MeSH
• Cell Proliferation * MeSH
• Flow Cytometry methods   MeSH
• In Vitro Techniques MeSH
• Tumor Stem Cell Assay methods   MeSH
• Cell Survival MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Publication type
• Journal Article MeSH
• Evaluation Study MeSH
• Names of Substances
• AC133 Antigen MeSH
• Hyaluronan Receptors MeSH
• Antigens, Neoplasm MeSH
• Antigens, CD MeSH
• Glycoproteins MeSH
• Integrin alpha6 MeSH
• Cell Adhesion Molecules MeSH
• Biomarkers, Tumor MeSH
• Peptides MeSH
• PROM1 protein, human MeSH Browser
• TACSTD2 protein, human MeSH Browser

PURPOSE: The purpose of this study was to investigate the expression of cytokeratin (CK) 8 in the corneoconjunctival epithelium. METHODS: In 17 cadaveric corneoscleral discs and 3 other discs, the presence of CK8 alone or CK8, together with CK3, CK15, vimentin, and integrin α6, was investigated by using indirect immunohistochemistry on radial cryosections. Four corneoscleral discs stored in organ culture were used for the preparation of tangential sections of the limbus and for the isolation of limbal epithelial cells and their subsequent cultivation. CK8 expression was examined by RT-PCR in the corneal, limbal, and conjunctival epithelium. RESULTS: Sixty percent of the cadaveric corneoscleral samples and all samples stored in organ culture revealed positivity for CK8 in the basal epithelial layer of the limbus. Positive basal cells formed a single line or separated clusters. The signal for CK8 became weaker toward the surface of the limbal epithelium. The colocalization of CK8 with vimentin and CK15 in the limbus was also found. CK3 showed only occasional positivity in some of the surface limbal cells. The expression of integrin α6 in the basal membrane was absent or decreased under the CK8-positive clusters. Cell cultures revealed strong positivity for CK8 in approximately 80% of the cultured cells, and CK8 expression in the cornea, limbus, and conjunctiva was determined by RT-PCR. CONCLUSIONS: The study demonstrates the strong expression of CK8 in limbal epithelial basal cells, which is maintained during the differentiation and migration of the limbal cells toward the central corneal epithelium.

• MeSH
• Cell Culture Techniques MeSH
• Adult MeSH
• Epithelial Cells metabolism   MeSH
• Gene Expression MeSH
• Microscopy, Fluorescence MeSH
• Fluorescent Antibody Technique, Indirect MeSH
• Integrin alpha6 metabolism   MeSH
• Keratin-15 metabolism   MeSH
• Keratin-3 metabolism   MeSH
• Keratin-8 genetics   metabolism   MeSH
• Conjunctiva cytology   metabolism   MeSH
• Middle Aged MeSH
• Humans MeSH
• Limbus Corneae cytology   metabolism   MeSH
• RNA, Messenger genetics   MeSH
• Adolescent MeSH
• Organ Culture Techniques MeSH
• Reverse Transcriptase Polymerase Chain Reaction MeSH
• Epithelium, Corneal cytology   metabolism   MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Vimentin metabolism   MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Adolescent MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Integrin alpha6 MeSH
• Keratin-15 MeSH
• Keratin-3 MeSH
• Keratin-8 MeSH
• RNA, Messenger MeSH
• Vimentin MeSH