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
• Cell Cycle MeSH
• Kinetics MeSH
• Humans MeSH
• DNA Repair MeSH
• DNA Replication * MeSH
• S Phase MeSH
• Uracil-DNA Glycosidase * metabolism   MeSH
• Uracil metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Uracil-DNA Glycosidase * MeSH
• Uracil MeSH

Deoxycytidine analogues (dCas) are widely used for the treatment of malignant diseases. They are commonly inactivated by cytidine deaminase (CDD), or by deoxycytidine monophosphate deaminase (dCMP deaminase). Additional metabolic pathways, such as phosphorylation, can substantially contribute to their (in)activation. Here, a new technique for the analysis of these pathways in cells is described. It is based on the use of 5-ethynyl 2'-deoxycytidine (EdC) and its conversion to 5-ethynyl 2'-deoxyuridine (EdU). Its use was tested for the estimation of the role of CDD and dCMP deaminase in five cancer and four non-cancer cell lines. The technique provides the possibility to address the aggregated impact of cytidine transporters, CDD, dCMP deaminase, and deoxycytidine kinase on EdC metabolism. Using this technique, we developed a quick and cheap method for the identification of cell lines exhibiting a lack of CDD activity. The data showed that in contrast to the cancer cells, all the non-cancer cells used in the study exhibited low, if any, CDD content and their cytidine deaminase activity can be exclusively attributed to dCMP deaminase. The technique also confirmed the importance of deoxycytidine kinase for dCas metabolism and indicated that dCMP deaminase can be fundamental in dCas deamination as well as CDD. Moreover, the described technique provides the possibility to perform the simultaneous testing of cytotoxicity and DNA replication activity.

• MeSH
• Cytidine * metabolism   MeSH
• Cytidine Deaminase metabolism   MeSH
• DCMP Deaminase * MeSH
• Deoxycytidine MeSH
• Deoxycytidine Kinase genetics   metabolism   MeSH
• Metabolic Networks and Pathways MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Cytidine * MeSH
• Cytidine Deaminase MeSH
• DCMP Deaminase * MeSH
• Deoxycytidine MeSH
• Deoxycytidine Kinase MeSH

Base excision repair is one of the important DNA repair mechanisms in cells. The fundamental role in this complex process is played by DNA glycosylases. Here, we present a novel approach for the real-time measurement of uracil DNA glycosylase activity, which employs selected oligonucleotides immobilized on the surface of magnetic nanoparticles and Förster resonance energy transfer. We also show that the approach can be performed by surface plasmon resonance sensor technology. We demonstrate that the immobilization of oligonucleotides provides much more reliable data than the free oligonucleotides including molecular beacons. Moreover, our results show that the method provides the possibility to address the relationship between the efficiency of uracil DNA glycosylase activity and the arrangement of the used oligonucleotide probes. For instance, the introduction of the nick into oligonucleotide containing the target base (uracil) resulted in the substantial decrease of uracil DNA glycosylase activity of both the bacterial glycosylase and glycosylases naturally present in nuclear lysates.

• Keywords
• Förster resonance energy transfer, base excision repair, immobilized oligonucleotides, surface plasmon resonance, uracil DNA glycosylase,
• MeSH
• Cell Nucleus metabolism   MeSH
• Humans MeSH
• Magnetic Iron Oxide Nanoparticles MeSH
• Oligonucleotide Probes chemistry   metabolism   MeSH
• DNA Repair MeSH
• Fluorescence Resonance Energy Transfer MeSH
• Uracil-DNA Glycosidase metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Oligonucleotide Probes MeSH
• Uracil-DNA Glycosidase 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.

• Keywords
• DNA dyes, cell metabolism, cell quantification, enzymatic conversion of substrate,
• MeSH
• Biological Assay MeSH
• DNA analysis   chemistry   metabolism   MeSH
• Fluorescent Dyes * MeSH
• Cell Count methods   MeSH
• Cell Proliferation MeSH
• Cell Survival MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Comparative Study MeSH
• Names of Substances
• DNA MeSH
• Fluorescent Dyes * 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.

• Keywords
• cytocentrifugation, microscopy, sample processing, staining,
• MeSH
• Staining and Labeling methods   MeSH
• Centrifugation instrumentation   methods   MeSH
• Cytological Techniques instrumentation   methods   MeSH
• Humans MeSH
• Microscopy methods   MeSH
• Specimen Handling methods   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

Cell quantification is widely used in basic or applied research. The current sensitive methods of cell quantification are exclusively based on the analysis of non-fixed cells and do not allow the simultaneous detection of various cellular components. A fast, sensitive and cheap method of the quantification of fixed adherent cells is described here. It is based on the incubation of DAPI- or Hoechst 33342-stained cells in a solution containing SDS. The presence of SDS results in the quick de-staining of DNA and simultaneously, in an up-to-1,000-fold increase of the fluorescence intensity of the used dyes. This increase can be attributed to the micelle formation of SDS. The method is sufficiently sensitive to reveal around 50-70 human diploid cells. It is compatible with immunocytochemical detections, the detection of DNA replication and cell cycle analysis by image cytometry. The procedure was successfully tested for the analysis of cytotoxicity. The method is suitable for the quantification of cells exhibiting low metabolic activity including senescent cells. The developed procedure provides high linearity and the signal is high for at least 20 days at room temperature. Only around 90 to 120 minutes is required for the procedure's completion.

• MeSH
• Staining and Labeling methods   MeSH
• Cell Adhesion MeSH
• Cell Line MeSH
• Cell Cycle MeSH
• Cytophotometry methods   MeSH
• Diploidy * MeSH
• DNA analysis   chemistry   MeSH
• Sodium Dodecyl Sulfate chemistry   MeSH
• Fluorescent Dyes chemistry   MeSH
• HeLa Cells MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• Cell Count instrumentation   methods   MeSH
• DNA Replication * MeSH
• Reproducibility of Results MeSH
• Cell Survival MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA MeSH
• Sodium Dodecyl Sulfate MeSH
• Fluorescent Dyes 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.

• Keywords
• click chemistry, indirect immunocytochemistry, isotopes, nucleoside and nucleotide analogues,
• MeSH
• Click Chemistry MeSH
• DNA chemistry   genetics   MeSH
• Halogenation MeSH
• In Situ Hybridization MeSH
• Immunohistochemistry MeSH
• Isotope Labeling MeSH
• Copper chemistry   MeSH
• Nucleosides chemistry   MeSH
• Nucleotides chemistry   MeSH
• Radioisotopes MeSH
• DNA Replication * MeSH
• Research MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• DNA MeSH
• Copper MeSH
• Nucleosides MeSH
• Nucleotides MeSH
• Radioisotopes MeSH