flow sorting
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Chromosome analysis and sorting using flow cytometry (flow cytogenetics) is an attractive tool for fractionating plant genomes to small parts. The reduction of complexity greatly simplifies genetics and genomics in plant species with large genomes. However, as flow cytometry requires liquid suspensions of particles, the lack of suitable protocols for preparation of solutions of intact chromosomes delayed the application of flow cytogenetics in plants. This chapter outlines a high-yielding procedure for preparation of solutions of intact mitotic chromosomes from root tips of young seedlings and for their analysis using flow cytometry and sorting. Root tips accumulated at metaphase are mildly fixed with formaldehyde, and solutions of intact chromosomes are prepared by mechanical homogenization. The advantages of the present approach include the use of seedlings, which are easy to handle, and the karyological stability of root meristems, which can be induced to high degree of metaphase synchrony. Chromosomes isolated according to this protocol have well-preserved morphology, withstand shearing forces during sorting, and their DNA is intact and suitable for a range of applications.
- MeSH
- buněčný cyklus MeSH
- chromozomy rostlin MeSH
- cytogenetika MeSH
- DNA rostlinná genetika MeSH
- hybridizace in situ fluorescenční metody MeSH
- karyotypizace MeSH
- meristém cytologie MeSH
- průtoková cytometrie metody MeSH
- rostlinné buňky MeSH
- rostliny genetika MeSH
- semena rostlinná růst a vývoj MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Many proteins are present in the nucleus; some are involved with its structural and functional organization, some with gene expression, and some with cell division. The plant nuclear proteome has not been well explored. Its characterization requires extraction methods which minimize both the artifactual alteration of the proteins and the extent of contamination with non-nuclear proteins. The conventional multi-step fractionation procedure is both laborious and prone to contamination. Here, we describe a single-step method based on flow sorting. The method allows the separation of G1, S and G2 phase nuclei and minimizes the risk of contamination by non-nuclear proteins. Preliminary results obtained using G1 phase cell nuclei from barley root tips indicate that flow sorting coupled with a protein/peptide separation and mass spectrometry will permit a comprehensive characterization of the plant nuclear proteome.
Flow cytometric analysis and sorting of plant mitotic chromosomes has been mastered by only a few laboratories worldwide. Yet, it has been contributing significantly to progress in plant genetics, including the production of genome assemblies and the cloning of important genes. The dissection of complex genomes by flow sorting into the individual chromosomes that represent small parts of the genome reduces DNA sample complexity and streamlines projects relying on molecular and genomic techniques. Whereas flow cytometric analysis, that is, chromosome classification according to fluorescence and light scatter properties, is an integral part of any chromosome sorting project, it has rarely been used on its own due to lower resolution and sensitivity as compared to other cytogenetic methods. To perform chromosome analysis and sorting, commercially available electrostatic droplet sorters are suitable. However, in order to resolve and purify chromosomes of interest the instrument must offer high resolution of optical signals as well as stability during long runs. The challenge is thus not the instrumentation, but the adequate sample preparation. The sample must be a suspension of intact mitotic metaphase chromosomes and the protocol, which includes the induction of cell cycle synchrony, accumulation of dividing cells at metaphase, and release of undamaged chromosomes, is time consuming and laborious and needs to be performed very carefully. Moreover, in addition to fluorescent staining chromosomal DNA, the protocol may include specific labelling of DNA repeats to facilitate discrimination of particular chromosomes. This review introduces the applications of chromosome sorting in plants, and discusses in detail sample preparation, chromosome analysis and sorting to achieve the highest purity in flow-sorted fractions, and their suitability for downstream applications.
These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer-reviewed by leading experts in the field, making this an essential research companion.
Cílem studie byla analýza prognostického významu p53, Ki-67, mitotického a apoptotického indexu a flow cytometrické analýzy karcinomů hlavy a krku. Materiál a metody: Hodnocen soubor 77 nemocných s karcinomem hlavy a krku léčených na Klinice otorinolaryngologie a chirurgie hlavy a krku v Brně v letech 2003–2005. Vedle obvyklých vyšetření byla provedena flow cytometrická analýza DNA a imunohistochemicky stanoven marker Ki-67 a p53. Výsledky: Se III. a IV. klinickým stadiem TNM klasifikace bylo léčeno 83,1 % nemocných. Histopatologický grading 1–2 mělo 77,9 % nemocných. Medián doby celkového přežití byl 39,7 měsíců. Vývoj rizika onemocnění během prvních 6 měsíců byl nezávislý na stagingu a lokalizaci. Málo pokročilé nádory (T1–2) a méně diferencované nádory (G3–4) měly lepší prognózu (p=0,034). Závěr: Hodnocení kinetických parametrů buněčného cyklu odhalilo významně nižší podíl buněk v S fázi u nádorů se špatnou prognózou. Nádory s přímou progresí ihned po léčbě dále vykazují statisticky vyšší podíl buněk v G2/M fázi.
The objective of the study was to analyze prognostic importance of the biomarkers p53, Ki67, mitotic and apoptotic index and flow cytometry analysis of the cell cycle of head and neck cancer in relation to Event Free Survival (EFS) and Overall Survival (OS). Material and Methods: the analysis was made in a group of 77 patients with head and neck cancer, who were treated at the Clinic of Otorhinolaryngology and Head and Neck Surgery at Brno in the years 2003–2005. In addition to common types of examinations (determination of localization, extent and possible metastases of the tumor histological examination), flow cytometric analysis of DNA was performed and the Ki-67 and p53 markers were examined by immunohistochemistry. Results: the tumor was localized in 31.2% of patients in nasopharynx and on the lateral wall of oropharynx, in 58.4% on the tongue root and in supraglottis and in 10.4% in hypopharynx. The IIIrd and IVth clinical stage of TNM classification was encoundeted in 83.1% of the treated patients. Histopathological grading 1 or 2 was established in 77.9% of patients. The median survival was 39.7 months. The evolution of the disease risk during the first 6 months appears to be independent of common clinical categories, specifically on staging and localization. The prognosis of low-advanced tumors (T1–2) and simultaneous low-differentiated tumors (G3–4) proved to be better (p=0.034). The prognosis of diploid and p53 negative tumors was better (p=0.222). Conclusions: the evaluation of kinetic parameters of the cell cycle univocally revealed a significantly lower proportion of cells in the S phase (named SPF) in tumor of poor prognosis. Tumors with a direct progression immediately after the therapy included a significantly higher proportion the cells in G2/M phase. The definition of poor or good prognosis is based on the ratio of SPF and G1/M phase.
- MeSH
- antigen Ki-67 diagnostické užití MeSH
- apoptóza MeSH
- biomedicínský výzkum MeSH
- buněčný cyklus účinky léků účinky záření MeSH
- finanční podpora výzkumu jako téma MeSH
- lidé MeSH
- mitotický index využití MeSH
- nádorový supresorový protein p53 diagnostické užití MeSH
- nádory hlavy a krku diagnóza klasifikace MeSH
- prognóza MeSH
- průtoková cytometrie využití MeSH
- retrospektivní studie MeSH
- Check Tag
- lidé MeSH
- Geografické názvy
- Česká republika MeSH
Nuclear genomes of many important plant species are tremendously complicated to map and sequence. The ability to isolate single chromosomes, which represent small units of nuclear genome, is priceless in many areas of plant research including cytogenetics, genomics, and proteomics. Flow cytometry is the only technique which can provide large quantities of pure chromosome fractions suitable for downstream applications including physical mapping, preparation of chromosome-specific BAC libraries, sequencing, and optical mapping. Here, we describe step-by-step procedure of preparation of liquid suspensions of intact mitotic metaphase chromosomes and their flow cytometric analysis and sorting.
BACKGROUND INFORMATION: Macarpine (MA) is a quaternary benzophenanthridine plant alkaloid isolated from Macleaya microcarpa or Stylophorum lasiocarpum. Benzophenanthridine alkaloids are interesting natural products that display antiproliferative, antimicrobial, antifungal and anti-inflammatory activities, and also fluorescence properties. In a previous study, we demonstrated that thanks to its ability to interact with DNA and its spectral properties MA could be used as a supravital DNA probe for fluorescence microscopy and flow cytometry including analyses of the cell cycle. In this study, we evaluated the suitability of MA as a DNA dye for time-lapse microscopy and flow-cytometric cell sorting. RESULTS: Living A-375 and MEF cells stained with MA were monitored by time-lapse microscopy for 24 h. Mitoses were observed at MA concentrations up to 0.5 μg/ml during the first 2-3 h. After this period of time, cells treated with MA at concentrations of 0.75 and 0.5 μg/ml underwent apoptosis. Cells cultivated with MA at concentration of 0.25 μg/ml or lower survived throughout the 24 h period. Toxicity of MA was dependent on light wavelength and frequency of image capturing. The intensity of MA fluorescence decreased during the incubation. MA concentration of 0.1 μg/ml was identified as the most suitable for live cell imaging with respect to fluorescence intensity and toxicity. MA at the concentration 10 μg/ml was used for sorting of enhanced green fluorescent protein (EGFP)-labelled neurons and fibroblasts yielding profiles similar to those obtained with DRAQ5. Contrary to DRAQ5, MA-stained cells survived in culture, and the sorted cells lost the MA signal suggesting reversible binding of the dye to the DNA. CONCLUSION: The results proved that MA may readily be used for chromosomes depicting and mitosis monitoring by time-lapse microscopy. In addition, MA has shown to be a suitable probe for sorting of EGFP-labelled cells, including neurons, that survived the labelling process. SIGNIFICANCE: In consideration of the results, we highly anticipate an onward use of MA in a broad range of applications based on live cell sorting and imaging, for example, cell synchronisation and monitoring of proliferation as an important experimental and/or diagnostic utility.
- MeSH
- benzofenantridiny analýza MeSH
- buněčné kultury MeSH
- buněčný cyklus fyziologie MeSH
- DNA analýza MeSH
- fluorescenční barviva analýza MeSH
- fluorescenční mikroskopie metody MeSH
- lidé MeSH
- průtoková cytometrie * metody MeSH
- separace buněk metody MeSH
- viabilita buněk MeSH
- zelené fluorescenční proteiny metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
