single-cell detection Dotaz Zobrazit nápovědu
Noninvasive metabolic assessment of single cells / David K. Gardner -- Laser capture microdissection for analysis of single cells / Nobuki Nakamura ... Scriven and Caroline Mackie Ogilvie -- Single cell PCR for PGD : methods, strategies, and limitations from single cells / Kenneth E. and detection of aneuploidy in single cells using DNA array-based comparative genomic hybridization
Methods in molecular medicine, ISSN 1543-1894 132
xi, 182 s. : il., tab.
Závěrečná zpráva o řešení grantu Agentury pro zdravotnický výzkum MZ ČR
nestr.
The proposed project builds on our continuous well-established research on chronic lymphocytic leukemia (CLL). In this study, we would like to profound our knowledge to better understand the mechanisms underlying clonal evolution of CLL cells during the disease course. We will employ a challenging approach a single cell analysis (SCA) for detailed characterisation of malignant cells on single cell level with aim to monitor disease progression and to detect the most aggressive subclones of CLL. In particular, we will analyse and compare transcriptomes of thousands of CLL cells from consecutively collected samples of patients suffering from early relapsed/refractory disease (R/R CLL), which is defined by non-response to treatment or relapse within six months after therapy. Thus, R/R CLL represents a highly challenging subtype of disorder with very poor prognosis and deserves further attention. A deeper understanding of molecular mechanisms driving R/R CLL can help to select the best treatment approach, especially from the growing spectrum of targeted therapy.
Navrhovaný projekt staví na dlouhodobém výzkumu naší skupiny v oblasti chronické lymfocytární leukémie (CLL). Projekt si klade za cíl prohloubit naše znalosti a lépe porozumět mechanismům klonální evoluce CLL buněk v průběhu onemocnění. Pro detailní charakterizaci nádorových buněk a sledování agresivních klonů v průběhu progrese onemocnění chceme využít moderní přístup analýzy na úrovni jednotlivých buněk (SCA). Budeme srovnávat transkriptomy tisíců CLL buněk získaných z opakovaných odběrů pacientů s relaps/refrakterní CLL (R/R CLL) což je onemocnění, které neodpovídá na léčbu nebo u něho dochází k progresi/relapsu dříve než za šest měsíců. R/R CLL reprezentuje významnou podskupinu onemocnění, která si zaslouží pozornost vzhledem k špatné prognóze onemocnění. Porozumění molekulárním mechanismům vedoucím k rozvoji R/R CLL umožní výběr nejvhodnější léčebné strategie, zvlášť v souvislosti s rychlým vývojem cílené léčby v poslední době.
- Klíčová slova
- klonální evoluce, chronic lymphocytic leukemia, clonal evolution, Chronická lymfocytární leukémie, single cell analysis, Single cell analýza,
- NLK Publikační typ
- závěrečné zprávy o řešení grantu AZV MZ ČR
Circulating tumor cells (CTCs) are rare cells that can be found in the peripheral blood of cancer patients. They have been demonstrated to be useful prognostic markers in many cancer types. Within the last decade various methods have been developed to detect rare cells within a liquid biopsy from a cancer patient. These methods have revealed the phenotypic diversity of CTCs and how they can represent the complement of cells that are found in a tumor. Single-cell proteogenomics has emerged as an all-encompassing next-generation technological approach for CTC research. This allows for the deconstruction of cellular heterogeneity, dynamics of metastatic initiation and progression, and response or resistance to therapeutics in the clinical settings. We take advantage of this opportunity to investigate CTC heterogeneity and understand their full potential in precision medicine.The high-definition single-cell analysis (HD-SCA) workflow combines detection of the entire population of CTCs and rare cancer related cells with single-cell genomic analysis and may therefore provide insight into their subpopulations based on molecular as well as morphological data. In this chapter we describe in detail the protocols from isolation of a candidate cell from a microscopy slide, through whole-genome amplification and library preparation, to CNV analysis of identified cells from the HD-SCA workflow. This process may also be applicable to any platform starting with a standard microscopy slide or isolated cell of interest.
DNA double stranded breaks (DSBs) are the most serious type of lesions introduced into chromatin by ionizing radiation. During DSB repair, cells recruit different proteins to the damaged sites in a manner dependent on local chromatin structure, DSB location in the nucleus, and the repair pathway entered. 53BP1 is one of the important players participating in repair pathway decision of the cell. Although many molecular biology details have been investigated, the architecture of 53BP1 repair foci and its development during the post-irradiation time, especially the period of protein recruitment, remains to be elucidated. Super-resolution light microscopy is a powerful new tool to approach such studies in 3D-conserved cell nuclei. Recently, we demonstrated the applicability of single molecule localization microscopy (SMLM) as one of these highly resolving methods for analyses of dynamic repair protein distribution and repair focus internal nano-architecture in intact cell nuclei. In the present study, we focused our investigation on 53BP1 foci in differently radio-resistant cell types, moderately radio-resistant neonatal human dermal fibroblasts (NHDF) and highly radio-resistant U87 glioblastoma cells, exposed to high-LET 15N-ion radiation. At given time points up to 24 h post irradiation with doses of 1.3 Gy and 4.0 Gy, the coordinates and spatial distribution of fluorescently tagged 53BP1 molecules was quantitatively evaluated at the resolution of 10⁻20 nm. Clusters of these tags were determined as sub-units of repair foci according to SMLM parameters. The formation and relaxation of such clusters was studied. The higher dose generated sufficient numbers of DNA breaks to compare the post-irradiation dynamics of 53BP1 during DSB processing for the cell types studied. A perpendicular (90°) irradiation scheme was used with the 4.0 Gy dose to achieve better separation of a relatively high number of particle tracks typically crossing each nucleus. For analyses along ion-tracks, the dose was reduced to 1.3 Gy and applied in combination with a sharp angle irradiation (10° relative to the cell plane). The results reveal a higher ratio of 53BP1 proteins recruited into SMLM defined clusters in fibroblasts as compared to U87 cells. Moreover, the speed of foci and thus cluster formation and relaxation also differed for the cell types. In both NHDF and U87 cells, a certain number of the detected and functionally relevant clusters remained persistent even 24 h post irradiation; however, the number of these clusters again varied for the cell types. Altogether, our findings indicate that repair cluster formation as determined by SMLM and the relaxation (i.e., the remaining 53BP1 tags no longer fulfill the cluster definition) is cell type dependent and may be functionally explained and correlated to cell specific radio-sensitivity. The present study demonstrates that SMLM is a highly appropriate method for investigations of spatiotemporal protein organization in cell nuclei and how it influences the cell decision for a particular repair pathway at a given DSB site.
Analysing the chemical content of individual cells has already been proven to reveal unique information on various biological processes. Single-cell analysis provides more accurate and reliable results for biology and medicine than analyses of extracts from cell populations, where a natural heterogeneity is averaged. To meet the requirements in the research of important biologically active molecules, such as caspases, we have developed a miniaturized device for simultaneous analyses of individual cells. A stainless steel body with a carousel holder enables high-sensitivity parallel detections in eight microvials. The holder is mounted in front of a photomultiplier tube with cooled photocathode working in photon counting mode. The detection of active caspase-3/7, central effector caspases in apoptosis, in single cells is based on the bioluminescence chemistry commercially available as Caspase-Glo®3/7 reagent developed by Promega. Individual cells were captured from a culture medium under microscope and transferred by micromanipulator into detection microvial filled with the reagent. As a result of testing, the limits of detection and quantification were determined to be 0.27/0.86 of active caspase-3/7 content in an average apoptotic cell and 0.46/2.92 for non-apoptotic cells. Application potential of this technology in laboratory diagnostics and related medical research is discussed. Graphical abstract Miniaturized device for simultaneous analyses of individual cells.
- MeSH
- analýza jednotlivých buněk přístrojové vybavení metody MeSH
- apoptóza * MeSH
- design vybavení MeSH
- enzymatické testy přístrojové vybavení metody MeSH
- kaspasa 3 analýza metabolismus MeSH
- kaspasa 7 analýza metabolismus MeSH
- kultivované buňky MeSH
- luminiscenční měření přístrojové vybavení metody MeSH
- myši MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Caspases are key enzymes activated during the apoptotic machinery. Apoptosis as a way of programmed cell death becomes deregulated in some pathologies including cancer transformations, neurodegenerative, or autoimmune diseases. Most of the methods available for the detection of apoptosis and caspases provide qualitative information only or quantification data as an average from cell populations or cell lysates. Several reports point to the importance of more accurate single-cell analyses in biomedical studies due to heterogeneity at tissue as well as cell level. To meet these requirements, we developed a miniaturized device enabling detection and quantification of active caspase-3/7 in individual cells at a femtogram level (10(-15) g). The active caspase-3/7 detection protocol is based on the bioluminescence chemistry commercially available as a Caspase-Glo™ 3/7 reagent developed by Promega. As a model, we used human stem cells treated by camptothecin to induce apoptosis. Individual apoptotic cells were captured from a culture medium under a microscope and transferred by a micromanipulation system into a detection capillary containing 2 μl of the reagent. Cells without activation by camptothecin served as negative controls. The detection limit of active caspase-3/7 achieved in the miniaturized system was determined as 0.20 and limit of quantification as 0.65 of the amount found in a single apoptotic human stem cell. Such a sensitive method could have a wide application potential in laboratory medicine and related clinically oriented research.
- MeSH
- analýza jednotlivých buněk přístrojové vybavení MeSH
- apoptóza * MeSH
- buněčná diferenciace MeSH
- crista neuralis cytologie MeSH
- design vybavení MeSH
- kamptothecin chemie MeSH
- kaspasa 3 metabolismus MeSH
- kaspasa 7 metabolismus MeSH
- kmenové buňky účinky léků patologie MeSH
- lidé MeSH
- luminiscence MeSH
- mikromanipulace MeSH
- miniaturizace přístrojové vybavení MeSH
- reprodukovatelnost výsledků MeSH
- zánět MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- MeSH
- fotoafinitní značky MeSH
- kathepsin D analýza MeSH
- lidé MeSH
- molekulární sondy chemie MeSH
- nádorové proteiny analýza MeSH
- nádory prsu chemie MeSH
- proteom MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Rapid and accurate identification of pathogens causing infections is one of the biggest challenges in medicine. Timely identification of causative agents and their antimicrobial resistance profile can significantly improve the management of infection, lower costs for healthcare, mitigate ever-growing antimicrobial resistance and in many cases, save lives. Raman spectroscopy was shown to be a useful-quick, non-invasive, and non-destructive -tool for identifying microbes from solid and liquid media. Modifications of Raman spectroscopy and/or pretreatment of samples allow single-cell analyses and identification of microbes from various samples. It was shown that those non-culture-based approaches could also detect antimicrobial resistance. Moreover, recent studies suggest that a combination of Raman spectroscopy with optical tweezers has the potential to identify microbes directly from human body fluids. This review aims to summarize recent advances in non-culture-based approaches of identification of microbes and their virulence factors, including antimicrobial resistance, using methods based on Raman spectroscopy in the context of possible use in the future point-of-care diagnostic process.
- MeSH
- analýza jednotlivých buněk MeSH
- antiinfekční látky * MeSH
- faktory virulence MeSH
- lidé MeSH
- Ramanova spektroskopie * metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
