Genome analysis in many plant species is hampered by large genome size and by sequence redundancy due to the presence of repetitive DNA and polyploidy. One solution is to reduce the sample complexity by dissecting the genomes to single chromosomes. This can be realized by flow cytometric sorting, which enables purification of chromosomes in large numbers. Coupling the chromosome sorting technology with next generation sequencing provides a targeted and cost effective way to tackle complex genomes. The methods outlined in this article describe a procedure for preparation of chromosomal DNA suitable for next-generation sequencing.
- MeSH
- Chromosomes, Plant ultrastructure MeSH
- Genome Size MeSH
- Microscopy, Fluorescence MeSH
- Genome, Plant * MeSH
- In Situ Hybridization, Fluorescence MeSH
- Hordeum cytology genetics MeSH
- Germination genetics MeSH
- Metaphase genetics MeSH
- Polyploidy MeSH
- Flow Cytometry methods MeSH
- Triticum cytology genetics MeSH
- Sequence Analysis, DNA MeSH
- Seeds genetics MeSH
- Chromosomes, Artificial, Bacterial MeSH
- High-Throughput Nucleotide Sequencing MeSH
- Secale cytology genetics MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- MeSH
- Biopsy methods MeSH
- DNA, Neoplasm analysis MeSH
- Carcinoma drug therapy genetics radiotherapy MeSH
- Humans MeSH
- Breast Neoplasms drug therapy genetics radiotherapy MeSH
- Flow Cytometry MeSH
- Check Tag
- Humans MeSH
- Publication type
- Comparative Study MeSH
Programmed cell death (PCD) is a crucial process required for the normal development and physiology of metazoans. The three major mechanisms that induce PCD are called type I (apoptosis), type II (autophagic cell death), and type III (necrotic cell death). Dysfunctional PCD leads to diseases such as cancer and neurodegeneration. Although apoptosis is the most common form of PCD, recent studies have provided evidence that there are other forms of cell death. The high majority of classical apoptotic hallmarks can be rapidly examined by flow cytometry. Cytometry thus became a technology of choice in diverse studies of cellular demise. A large variety of cytometric methods designed to identify apoptotic cells and probe mechanisms associated with this mode of cell demise have been developed during the past two decades.
Smíšená lymfocytární reakce (MLC) T-lymfocytů s dendritickými buňkami je jedním ze základních nástrojů studia imunologických mechanismů. Tato práce přináší popis nové metodiky hodnocení alogenní a autologní MLC pomocí průtokové cytometrie a diferenciálního gatingu. Metoda je založena na časově omezené akvizici událostí a na jejich rozdělení podle hodnot forward a side scatteru. Gatována je jednak populace všech živých buněk (R1), dále populace živých neproliferujících buněk (R2) a populace mrtvých/apoptotických buněk (R4). Pomocí fluorescenčních mikrosfér (R3) je navíc možno vypočítat absolutní množství buněk ve stanoveném gatu. Pomocí 7-AAD exkluze bylo ověřeno, že zhruba 90 % 7-AAD pozitivních buněk se promítá do gatu R4, navíc poměr R1: R2 výtečně koreluje s procentem buněk pozitivních na aktivační/proliferační marker CD71. Příklady dalších výsledků dosažených touto metodikou jsou uvedeny v textu. Vzhledem k tomu, že metoda diferenciálního gatingu ve své základní podobě nevyžaduje užití žádné fluorescenční protilátky, umožňuje rychlý a levný skríning velkého množství vzorků bez použití radioaktivně značených nukleotidů.
Mixed lymphocyte reaction (MLC) of T-lymphocytes with dendritic cells is one of the basic tools for studying of immune reaction mechanisms. This work describes a new method of evaluation of allogeneic and autologous MLC by flow cytometry and differential gating. This method is based on fixed time acquisition of events and their sorting according to their forward and side scatter properties. Differential gating distinguishes populations of all living cells (R1), living nonproliferating cells (R2) and dead/apoptotic cells (R4). Addition of fluorescence microspheres (R3) enables the calculation of absolute number of cells in a given sample volume. Using the method of 7-AAD exclusion, we have shown that approximately 90% of 7-AAD positive cells project into the R4 gate. Moreover, there was an excellent correlation between the R1: R2 ratio and the percentage of cells positive for activation/proliferation marker CD71. The method of differential gating in its basic version does not require use of any fluorescent antibody and therefore is suitable for rapid screening of large number of samples at reasonable cost and without the use of radiolabeled nucleotides.
Imunofenotypizace myelomových buněk přináší dostatek informací k charakterizaci a odlišení těchto heterogenních neoplastických plazmocytů od jejich normálních protějšků a své uplatnění tedy nalézá v diferenciální diagnostice. Stanovení antigenního profilu PC může též napomoci identifikaci prognostických markerů, které by vedly jednak k jednoznačnému stanovení diagnózy a určení pravděpodobnosti progrese u asymptomatických monoklonálních gamapatií, ale také k případnému hodnocení minimální reziduální choroby či účinnosti léčby u mnohočetného myelomu. Je snahou Evropské myelomové sítě (European Myeloma Network), EMN, sjednotit a standardizovat fl owcytometrické vyšetření plazmocytů v rámci monoklonálních gamapatií.
Immunophenotyping of myeloma cells is used to characterize the malignant plasma cells and to distinguish them from their normal counterparts. Flow cytometry is mostly used for the diagnosis of plasma cell proliferations but a detailed analysis of plasma cell immunophenotype can identify disease markers that are associated with the progression of monoclonal gammopathy of unknown signifi cance to multiple myeloma (MM), the prognosis of MM, the response to treatment, and the presence of minimal residual disease. The aim of the European Myeloma Network (EMN) is to unify and standardize immunophenotyping methods for the analysis of plasma cells in monoclonal gammopathies.
- MeSH
- Immunophenotyping MeSH
- Bone Marrow MeSH
- Humans MeSH
- Multiple Myeloma * genetics physiopathology MeSH
- Plasma Cells * cytology MeSH
- Flow Cytometry * methods MeSH
- Check Tag
- Humans MeSH
- Publication type
- Research Support, Non-U.S. Gov't 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.
Sugarcane (Saccharum spp.) is a globally important crop for sugar and bioenergy production. Its highly polyploid, complex genome has hindered progress in understanding its molecular structure. Flow cytometric sorting and analysis has been used in other important crops with large genomes to dissect the genome into component chromosomes. Here we present for the first time a method to prepare suspensions of intact sugarcane chromosomes for flow cytometric analysis and sorting. Flow karyotypes were generated for two S. officinarum and three hybrid cultivars. Five main peaks were identified and each genotype had a distinct flow karyotype profile. The flow karyotypes of S. officinarum were sharper and with more discrete peaks than the hybrids, this difference is probably due to the double genome structure of the hybrids. Simple Sequence Repeat (SSR) markers were used to determine that at least one allelic copy of each of the 10 basic chromosomes could be found in each peak for every genotype, except R570, suggesting that the peaks may represent ancestral Saccharum sub genomes. The ability to flow sort Saccharum chromosomes will allow us to isolate and analyse chromosomes of interest and further examine the structure and evolution of the sugarcane genome.
- MeSH
- Alleles MeSH
- Cell Cycle drug effects genetics MeSH
- Chromosomes, Plant genetics MeSH
- DNA, Plant metabolism MeSH
- Fluorescence MeSH
- Genome, Plant * MeSH
- Hydroxyurea pharmacology MeSH
- Karyotype MeSH
- Kinetics MeSH
- Plant Roots drug effects MeSH
- Polyploidy * MeSH
- Flow Cytometry methods MeSH
- Saccharum drug effects genetics MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Východiska: Vícebarevná průtoková cytometrie je užitečným nástrojem k analýze plazmatických buněk, jelikož s dostatečnou senzitivitou dovoluje stanovení jejich počtu a zejména rozlišení na fyziologické a patologické. Typ studie a soubor: V této studii jsme sledovali zastoupení plazmocytů na souboru 25 pacientů s monoklonální gamapatií nejasného významu a 78 osob s mnohočetným myelomem, s cílem nalézt rozdíly v počtu a fenotypu jejich plazmocytů a případně také defi novat potenciální parametr, který by obě nemoci od sebe odlišil. Metody a výsledky: Flowcytometrická analýza plazmocytů byla prováděna ve vzorcích kostních dření s využitím povrchových markerů CD38, CD138, CD45, CD56 a CD19. Při srovnání s mnohočetným myelomem je pro monoklonální gamapatii nejasného významu příznačné vyšší zastoupení reziduálních CD19+ plazmocytů (p<0,001) a nižší zastoupení patologických CD56+ plazmocytů (p=0,004) v kostní dřeni. Závěry: Relativní počet CD19+ plazmocytů se jeví jako jednoznačný parametr pro odlišení monoklonální gamapatie nejasného významu od všech klinických stádií mnohočetného myelomu. Analýzou plazmocytů a jejich fenotypového profi lu můžeme napomoci při diferenciální diagnostice onemocnění. Do budoucna předpokládáme také možný význam analýzy plazmocytů v kostní dřeni pro prognózu hodnocených osob a pro studium minimální reziduální choroby.
Backgrounds: Multicolor fl ow cytometry is a sensitive tool for the analysis of plasma cell population. It can differentiate between normal and malignant plasma cells and enables their quantifi cation. Design and Subjects: We have used fl ow cytometry for quantifi cation and immunophenotyping of plasma cells from 25 patients with monoclonal gammopathy of undetermined signifi cance and 78 those with multiple myeloma aiming to identify and characterize markers that distinguish between the two conditions. Methods and Results: The expression of surface antigens CD38, CD138, CD45, CD56 and CD19 was analysed on bone marrow plasma cells. As compared to MM patients, MGUS patients had higher proportion of residual CD19+ plasma cells (p<0,001) and lower counts of pathologic CD56+ plasma cells (p=0,004) in the bone marrow. Conclusions: The number of CD19+ plasma cells appeared to be an unequivocal marker that distinguishes monoclonal gammopathy of unknown signifi cance from all stages of multiple myeloma. Immunophenotyping of plasma cell can thus contribute to the differential diagnosis of both diseases. It has also been suggested that flow cytometric analysis should be useful for prognostic evaluation and for the study of minimal residual disease.
- MeSH
- CD56 Antigen MeSH
- Antigens, CD19 MeSH
- Gene Expression MeSH
- Phenotype MeSH
- Bone Marrow MeSH
- Multiple Myeloma genetics pathology MeSH
- Monoclonal Gammopathy of Undetermined Significance genetics pathology MeSH
- Antibodies, Monoclonal MeSH
- Plasma Cells * cytology physiology pathology MeSH
- Flow Cytometry * utilization MeSH
- Sensitivity and Specificity MeSH
- Severity of Illness Index MeSH
- Research Design MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
