The distribution of fluorescence signals measured with flow cytometry can be influenced by several factors, including qualitative and quantitative properties of the used fluorochromes, optical properties of the detection system, as well as the variability within the analyzed cell population itself. Most of the single cell samples prepared from in vitrocultures or clinical specimens contain a variable cell cycle component. Cell cycle, together with changes in the cell size, are two of the factors that alter the functional properties of analyzed cells and thus affect the interpretation of obtained results. Here, we describe the association between cell cycle status and cell size, and the variability in the distribution of fluorescence intensity as determined with flow cytometry, at population scale. We show that variability in the distribution of background and specific fluorescence signals is related to the cell cycle state of the selected population, with the 10% low fluorescence signal fraction enriched mainly in cells in their G0/G1 cell cycle phase, and the 10% high fraction containing cells mostly in the G2/M phase. Therefore we advise using caution and additional experimental validation when comparing populations defined by fractions at both ends of fluorescence signal distribution to avoid biases caused by the effect of cell cycle and cell size.
- MeSH
- buněčné dělení MeSH
- buněčný cyklus fyziologie MeSH
- G2 fáze * MeSH
- průtoková cytometrie metody MeSH
- velikost buňky MeSH
- Publikační typ
- časopisecké články MeSH
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
- antigeny CD44 metabolismus MeSH
- antigeny nádorové metabolismus MeSH
- CD antigeny metabolismus MeSH
- glykoproteiny metabolismus MeSH
- integrin alfa6 metabolismus MeSH
- lidé MeSH
- molekuly buněčné adheze metabolismus MeSH
- nádorové biomarkery metabolismus MeSH
- nádorové buněčné linie MeSH
- nádorové kmenové buňky metabolismus patologie MeSH
- nádory prostaty metabolismus patologie MeSH
- nádory tračníku metabolismus patologie MeSH
- peptidy metabolismus MeSH
- proliferace buněk * MeSH
- průtoková cytometrie metody MeSH
- techniky in vitro MeSH
- testy nádorových kmenových buněk metody MeSH
- viabilita buněk MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
