Mismatched nucleobase uracil is commonly repaired through the base excision repair initiated by DNA uracil glycosylases. The data presented in this study strongly indicate that the nuclear uracil-N-glycosylase activity and nuclear protein content in human cell lines is highest in the S phase of the cell cycle and that its distribution kinetics partially reflect the DNA replication activity in replication foci. In this respect, the data demonstrate structural changes of the replication focus related to the uracil-N-glycosylase distribution several dozens of minutes before end of its replication. The analysis also showed that very popular synchronisation protocols based on the double thymidine block can result in changes in the UNG2 content and uracil excision rate. In response, we propose a new method for the description of the changes of the content and the activity of different cell components during cell cycle without the necessity to use synchronisation protocols.
- MeSH
- buněčný cyklus MeSH
- kinetika MeSH
- lidé MeSH
- oprava DNA MeSH
- replikace DNA * MeSH
- S fáze MeSH
- uracil-DNA-glykosidasa * metabolismus MeSH
- uracil metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- uracil-DNA-glykosidasa * MeSH
- uracil MeSH
Cellular growth and the preparation of cells for division between two successive cell divisions is called the cell cycle. The cell cycle is divided into several phases; the length of these particular cell cycle phases is an important characteristic of cell life. The progression of cells through these phases is a highly orchestrated process governed by endogenous and exogenous factors. For the elucidation of the role of these factors, including pathological aspects, various methods have been developed. Among these methods, those focused on the analysis of the duration of distinct cell cycle phases play important role. The main aim of this review is to guide the readers through the basic methods of the determination of cell cycle phases and estimation of their length, with a focus on the effectiveness and reproducibility of the described methods.
- Klíčová slova
- BrdU, DNA labeling, EdU, cell cycle, labeled nucleosides, markers of cell cycle phases, time lapse microscopy,
- MeSH
- bromodeoxyuridin * metabolismus MeSH
- buněčné dělení MeSH
- buněčný cyklus MeSH
- proliferace buněk MeSH
- reprodukovatelnost výsledků MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
- Názvy látek
- bromodeoxyuridin * MeSH
Cell quantification is widely used both in basic and applied research. A typical example of its use is drug discovery research. Presently, plenty of methods for cell quantification are available. In this review, the basic techniques used for cell quantification, with a special emphasis on techniques based on fluorescent DNA dyes, are described. The main aim of this review is to guide readers through the possibilities of cell quantification with various methods and to show the strengths and weaknesses of these methods, especially with respect to their sensitivity, accuracy, and length. As these methods are frequently accompanied by an analysis of cell proliferation and cell viability, some of these approaches are also described.
- Klíčová slova
- DNA dyes, cell metabolism, cell quantification, enzymatic conversion of substrate,
- MeSH
- biotest MeSH
- DNA analýza chemie metabolismus MeSH
- fluorescenční barviva * MeSH
- počet buněk metody MeSH
- proliferace buněk MeSH
- viabilita buněk MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
- srovnávací studie MeSH
- Názvy látek
- DNA MeSH
- fluorescenční barviva * MeSH
Cytocentrifugation is a common technique for the capture of cells on microscopic slides. It usually requires a special cytocentrifuge or cytorotor and cassettes. In the study presented here, we tested the new concept of cytocentrifugation based on the threaded connection of the lid and the sample holder to ensure an adjustable flow of solutions through the filters and the collection of the filtered solutions in the reservoir during centrifugation. To test this concept, we developed a device for the preparation of cell samples on circular coverslips. The device was tested for the capture and sample processing of both eukaryotic and prokaryotic cells, cell nuclei, and mitochondria for microscopy analysis including image cytometry. Moreover, an efficient procedure was developed for capturing formaldehyde-fixed cells on non-treated coverslips without cell drying. The results showed that the tested arrangement enables the effective capture and processing of all of the tested samples and the developed device represents an inexpensive alternative to common cytocentrifuges, as only the paper filter is consumed during sample processing, and no special centrifuge, cytorotor, or cassette is necessary. As no additional system of solution removal is required during sample staining, the tested concept also facilitates the eventual automation of the staining procedure.
- Klíčová slova
- cytocentrifugation, microscopy, sample processing, staining,
- MeSH
- barvení a značení metody MeSH
- centrifugace přístrojové vybavení metody MeSH
- cytologické techniky přístrojové vybavení metody MeSH
- lidé MeSH
- mikroskopie metody MeSH
- odběr biologického vzorku metody MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
The replication of nuclear and mitochondrial DNA are basic processes assuring the doubling of the genetic information of eukaryotic cells. In research of the basic principles of DNA replication, and also in the studies focused on the cell cycle, an important role is played by artificially-prepared nucleoside and nucleotide analogues that serve as markers of newly synthesized DNA. These analogues are incorporated into the DNA during DNA replication, and are subsequently visualized. Several methods are used for their detection, including the highly popular click chemistry. This review aims to provide the readers with basic information about the various possibilities of the detection of replication activity using nucleoside and nucleotide analogues, and to show the strengths and weaknesses of those different detection systems, including click chemistry for microscopic studies.
- Klíčová slova
- click chemistry, indirect immunocytochemistry, isotopes, nucleoside and nucleotide analogues,
- MeSH
- click chemie MeSH
- DNA chemie genetika MeSH
- halogenace MeSH
- hybridizace in situ MeSH
- imunohistochemie MeSH
- izotopové značení MeSH
- měď chemie MeSH
- nukleosidy chemie MeSH
- nukleotidy chemie MeSH
- radionuklidy MeSH
- replikace DNA * MeSH
- výzkum MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
- Názvy látek
- DNA MeSH
- měď MeSH
- nukleosidy MeSH
- nukleotidy MeSH
- radionuklidy MeSH
