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
- Cell Cycle MeSH
- Chromosomes, Plant * MeSH
- Cytogenetics MeSH
- DNA, Plant genetics MeSH
- In Situ Hybridization, Fluorescence methods MeSH
- Karyotyping MeSH
- Meristem cytology MeSH
- Flow Cytometry methods MeSH
- Plant Cells MeSH
- Plants genetics MeSH
- Seeds growth & development MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- DNA, Plant 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.
- Keywords
- Cell cycle synchronization, Chromosome isolation, Cytogenetic stocks, FISH, FISHIS, Flow cytometry and sorting, Metaphase accumulation, Plants,
- MeSH
- Chromosomes, Plant * MeSH
- In Situ Hybridization, Fluorescence methods MeSH
- Flow Cytometry methods MeSH
- Plants genetics MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Flow cytometry offers a unique way of analyzing and manipulating plant chromosomes. During a rapid movement in a liquid stream, large populations can be classified in a short time according to their fluorescence and light scatter properties. Chromosomes whose optical properties differ from other chromosomes in a karyotype can be purified by flow sorting and used in a range of applications in cytogenetics, molecular biology, genomics, and proteomics. As the samples for flow cytometry must be liquid suspensions of single particles, intact chromosomes must be released from mitotic cells. This protocol describes a procedure for preparation of suspensions of mitotic metaphase chromosomes from meristem root tips and their flow cytometric analysis and sorting for various downstream applications.
- Keywords
- Accumulation of metaphase cells, Chromosome isolation, Cytogenetic stocks, FISH, FISHIS, Flow cytometry and sorting, Hydroponic, Mitotic synchrony, Plants, Seedlings,
- MeSH
- Chromosomes, Plant * MeSH
- Chromosomes * MeSH
- Cytogenetics MeSH
- Karyotyping MeSH
- Flow Cytometry methods MeSH
- Suspensions MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Suspensions MeSH
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.
Analysis and sorting of plant chromosomes (plant flow cytogenetics) is a special application of flow cytometry in plant genomics and its success depends critically on sample quality. This unit describes the methodology in a stepwise manner, starting with the induction of cell cycle synchrony and accumulation of dividing cells in mitotic metaphase, and continues with the preparation of suspensions of intact mitotic chromosomes, flow analysis and sorting of chromosomes, and finally processing of the sorted chromosomes. Each step of the protocol is described in detail as some procedures have not been used widely. Supporting histograms are presented as well as hints on dealing with plant material; the utility of sorted chromosomes for plant genomics is also discussed. © 2016 by John Wiley & Sons, Inc.
- Keywords
- cell cycle synchronization, chromosome genomics, chromosome isolation, chromosome sorting, chromosome-specific DNA libraries, flow cytometry, genome sequencing, high-molecular-weight DNA, physical genome mapping, plant chromosomes,
- MeSH
- Chromosomes, Plant metabolism MeSH
- DNA, Plant genetics MeSH
- In Situ Hybridization, Fluorescence MeSH
- Karyotyping MeSH
- Meristem cytology drug effects MeSH
- Metaphase drug effects MeSH
- Molecular Weight MeSH
- Nitrous Oxide pharmacology MeSH
- Proteomics MeSH
- Flow Cytometry methods MeSH
- Plants genetics MeSH
- Seeds drug effects MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- DNA, Plant MeSH
- Nitrous Oxide MeSH
Procedures for chromosome analysis and sorting using flow cytometry (flow cytogenetics) were developed for rye (Secale cereale L.). Suspensions of intact chromosomes were prepared by mechanical homogenization of synchronized root tips after mild fixation with formaldehyde. Histograms of relative fluorescence intensity obtained after the analysis of DAPI-stained chromosomes (flow karyotypes) were characterized and the chromosome content of the DNA peaks was determined. Chromosome 1R could be discriminated on a flow karyotype of S. cereale 'Imperial'. The remaining rye chromosomes (2R-7R) could be discriminated and sorted from individual wheat-rye addition lines. The analysis of lines with reconstructed karyotypes demonstrated a possibility of sorting translocation chromosomes. Supernumerary B chromosomes could be sorted from an experimental rye population and from S. cereale 'Adams'. Flow-sorted chromosomes were identified by fluorescence in situ hybridization (FISH) with probes for various DNA repeats. Large numbers of chromosomes of a single type sorted onto microscopic slides facilitated detection of rarely occurring chromosome variants by FISH with specific probes. PCR with chromosome-specific primers confirmed the identity of sorted fractions and indicated suitability of sorted chromosomes for physical mapping. The possibility to sort large numbers of chromosomes opens a way for the construction of large-insert chromosome-specific DNA libraries in rye.
- MeSH
- Chromosomes, Plant genetics MeSH
- Physical Chromosome Mapping MeSH
- In Situ Hybridization, Fluorescence MeSH
- Karyotyping MeSH
- Microsatellite Repeats MeSH
- Polymerase Chain Reaction MeSH
- Flow Cytometry methods MeSH
- Cell Separation methods MeSH
- Translocation, Genetic MeSH
- Secale genetics MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Optical mapping-a technique that visualizes short sequence motives along DNA molecules of hundred kilobases to megabase in size-has found an important place in genome research. It is widely used to facilitate genome sequence assemblies and analyses of genome structural variations. Application of the technique is conditional on availability of highly pure ultra-long high-molecular-weight DNA (uHMW DNA), which is challenging to achieve in plants due to the presence of the cell wall, chloroplasts, and secondary metabolites, just as a high content of polysaccharides and DNA nucleases in some species. These obstacles can be overcome by employment of flow cytometry, enabling a fast and highly efficient purification of cell nuclei or metaphase chromosomes, which are afterward embedded in agarose plugs and used to isolate the uHMW DNA in situ. Here, we provide a detailed protocol for the flow sorting-assisted uHMW DNA preparation that has been successfully used to construct whole-genome as well as chromosomal optical maps for 20 plant species from several plant families.
- Keywords
- Bionano genome map, Chromosomes, Flow cytometry and sorting, HMW DNA preparation, Nuclei, Optical mapping, ultralong high-molecular-weight DNA,
- MeSH
- Chromosomes, Plant * genetics MeSH
- Genome, Plant MeSH
- Flow Cytometry methods MeSH
- Restriction Mapping MeSH
- Plants * genetics MeSH
- Sequence Analysis, DNA methods MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Antibody reagents are the key components of multiparametric flow cytometry analysis. Their quality performance is an absolute requirement for reproducible flow cytometry experiments. While there is an enormous body of antibody reagents available, there is still a lack of consensus about which criteria should be evaluated to select antibody reagents with the proper performance, how to validate antibody reagents for flow cytometry, and how to interpret the validation results. The achievements of cytometry moved the field to a higher number of measured parameters, large data sets, and computational data analysis approaches. These advancements pose an increased demand for antibody reagent performance quality. This review summarizes the codevelopment of cytometry, antibody development, and validation strategies. It discusses the diverse issues of the specificity, cross-reactivity, epitope, titration, and reproducibility features of antibody reagents, and this review discusses the validation principles and methods that are currently available and those that are emerging. We argue that significant efforts should be invested by antibody users, developers, manufacturers, and publishers to increase the quality and reproducibility of published studies. More validation data should be presented by all stakeholders; however, the data should be presented in sufficient experimental detail to foster reproducibility, and community effort shall lead to the public availability of large data sets that can serve as a benchmark for antibody performance. © 2019 International Society for Advancement of Cytometry.
- Keywords
- flow cytometry, monoclonal antibody, validation,
- MeSH
- Indicators and Reagents MeSH
- Antibodies * MeSH
- Flow Cytometry MeSH
- Reproducibility of Results MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Review MeSH
- Names of Substances
- Indicators and Reagents MeSH
- Antibodies * MeSH
The use of flow cytometry for evaluation of plant chromosomes requires some specialized attention to preparation and instrumentation. This unit deals exclusively with plant cytogenetics and presents an outline of this area as well as methods for accumulation of cells in metaphase, preparation of chromosome suspensions, flow analysis and sorting of chromosomes, and processing of the sorted chromosomes. Each method is described in tremendous detail because in many aspects dealing with plant cells is quite different from dealing with mammalian cells. Supporting histograms are presented as well as a range of special hints on dealing with plant material and a discussion of the utility of sorted chromosomes for plant genome mapping.
- MeSH
- Cell Cycle MeSH
- Chromosomes, Plant chemistry genetics MeSH
- Chromosomes ultrastructure MeSH
- Cytogenetics MeSH
- Cytological Techniques methods MeSH
- DNA, Plant genetics MeSH
- Physical Chromosome Mapping MeSH
- Genome MeSH
- Gene Library MeSH
- Flow Cytometry methods MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- DNA, Plant MeSH
The Flow Cytometry Standard (FCS) format is a widely accepted norm for storing Flow Cytometry (FCM) data. Its goal as a standard is to allow FCM data sharing and re-analysis. Over more than three decades of its existence FCS has evolved into a well-defined, flexible file format reflecting technical changes in the FCM field. Its flexibility as well as rising numbers of instrument vendors leads to suboptimal implementations of FCS in some cases. Such situations compromise the primary goal of the standard and hinder the ability to reproduce FCM analyses. It is further underlined by rapid rise of advanced FCM analyses, often carried out outside traditional software tools and heavily relying on standard data storage and presentation. We have developed flowIO, an R package which tests FCS file conformance with the standard as defined by International Society for Advancement of Cytometry (ISAC) normative. Along with the package we provide a web based application (also at http://bioinformin.cesnet.cz/flowIO/) allowing user friendly access to the conformance testing as well as FCS file editing and export for further analysis.
- Keywords
- FCS, bioinformatics, data standard, file format, flow cytometry, reproducibility in science,
- MeSH
- Flow Cytometry standards MeSH
- Software * MeSH
- Computational Biology * MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
