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

Contamination of cell cultures by mycoplasmas is a very common phenomenon. As they can substantially alter cell metabolism and potentially spread to all cell cultures in laboratory, their early detection is necessary. One of the fastest and cheapest methods of mycoplasma detection relies on the direct staining of mycoplasmas' DNA by DAPI or Hoechst dyes. Although this method is easy and fast to perform, it suffers from the low signal provided by these dyes compared to the nuclear DNA. Therefore, the reporter cell lines are used for cultivation of mycoplasmas before DAPI or the Hoechst staining step. In the study presented, we have developed and tested a new immunofluorescence assay for the detection of mycoplasmas. The method is based on the enzymatic labeling using DNA polymerase I and modified nucleotides utilizing nicks in the mycoplasmas' DNA. Modified nucleotides are incorporated into mycoplasmas' DNA and subsequently visualized by immunofluorescence microscopy. The developed approach is independent of the mycoplasma strain, does not intensely stain nuclear DNA, does not stain other bacteria, and provides higher sensitivity than the approach based on the direct labeling using DAPI or Hoechst dyes.

• Klíčová slova
• cell cultures, immunofluorescence detection, mycoplasma infection,
• MeSH
• barvení a značení MeSH
• buňky A549 MeSH
• DNA-polymerasa I chemie   MeSH
• fluorescenční mikroskopie metody   MeSH
• lidé MeSH
• Mycoplasma fermentans izolace a purifikace   MeSH
• Mycoplasma hominis izolace a purifikace   MeSH
• Mycoplasma izolace a purifikace   MeSH
• mykoplazmové infekce mikrobiologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA-polymerasa I MeSH

We have developed a simple system for the analysis of the affinity of anti-bromodeoxyuridine antibodies. The system is based on the anchored oligonucleotides containing 5-bromo-2'-deoxyuridine (BrdU) at three different positions. It allows a reliable estimation of the reactivity of particular clones of monoclonal anti-bromodeoxyuridine antibodies with BrdU in fixed and permeabilized cells. Using oligonucleotide probes and four different protocols for the detection of BrdU incorporated in cellular DNA, we identified two antibody clones that evinced sufficient reactivity to BrdU in all the tested protocols. One of these clones exhibited higher reactivity to 5-iodo-2'-deoxyuridine (IdU) than to BrdU. It allowed us to increase the sensitivity of the used protocols without a negative effect on the cell physiology as the cytotoxicity of IdU was comparable with BrdU and negligible when compared to 5-ethynyl-2'-deoxyuridine. The combination of IdU and the improved protocol for oxidative degradation of DNA provided a sensitive and reliable approach for the situations when the low degradation of DNA and high BrdU signal is a priority.

• MeSH
• bromodeoxyuridin metabolismus   MeSH
• buněčné klony MeSH
• DNA metabolismus   MeSH
• HCT116 buňky MeSH
• HeLa buňky MeSH
• idoxuridin analogy a deriváty   metabolismus   MeSH
• lidé MeSH
• monoklonální protilátky metabolismus   MeSH
• peptidové mapování * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 5'-deoxy-5'-iodouridine MeSH Prohlížeč
• bromodeoxyuridin MeSH
• DNA MeSH
• idoxuridin MeSH
• monoklonální protilátky MeSH