Single-cell workflow
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The limited availability of biological samples hinders phylogenetic efforts to define structural differences among various biological groups. A novel workflow enabling the analysis of protists in low cell numbers by electron microscopy (EM) is described with cysts of Giardia intestinalis, a single-celled eukaryotic parasite. Correlative light and electron microscopy (CLEM) allows for the selection of individual cells and is economical in terms of time and cost. We describe a cyst purification protocol in combination with an adhesive coating for fixation and ultrathin embedding that results in excellent preservation of cell morphology. The application of advanced structural and analytical EM methods, such as high-resolution field emission scanning electron microscopy (FESEM), focused ion beam tomography (FIB/SEM), and energy-dispersive X-ray spectroscopy (EDX) analysis, is demonstrated. The workflow represents a new approach for studying the cellular and organelle architecture of rare and "difficult to culture" microorganisms.
As cancer care is transitioning to personalized therapies with necessary complementary or companion biomarkers there is significant interest in determining to what extent non-invasive liquid biopsies reflect the gold standard solid biopsy. We have established an approach for measuring patient-specific circulating and solid cell concordance by introducing tumor touch preparations to the High-Definition Single Cell Analysis workflow for high-resolution cytomorphometric characterization of metastatic colorectal cancer (mCRC). Subgroups of cells based on size, shape and protein expression were identified in both liquid and solid biopsies, which overall displayed high inter- and intra- patient pleomorphism at the single-cell level of analysis. Concordance of liquid and solid biopsies was patient-dependent and between 0.1-0.9. Morphometric variables displayed particularly high correlation, suggesting that circulating cells do not represent distinct subpopulations from the solid tumor. This was further substantiated by significant decrease in concentration of circulating cells after mCRC resection. Combined with the association of circulating cells with tumor burden and necrosis of hepatic lesions, our overall findings demonstrate that liquid biopsy cells can be informative biomarkers in the mCRC setting. Patient-specific level of concordance can readily be measured to establish the utility of circulating cells as biomarkers and define biosignatures for liquid biopsy assays.
- Publikační typ
- časopisecké články MeSH
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.
SUMMARY: ShinySOM offers a user-friendly interface for reproducible, high-throughput analysis of high-dimensional flow and mass cytometry data guided by self-organizing maps. The software implements a FlowSOM-style workflow, with improvements in performance, visualizations and data dissection possibilities. The outputs of the analysis include precise statistical information about the dissected samples, and R-compatible metadata useful for the batch processing of large sample volumes. AVAILABILITY AND IMPLEMENTATION: ShinySOM is free and open-source, available online at gitlab.com/exaexa/ShinySOM. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
- MeSH
- algoritmy * MeSH
- metadata MeSH
- průběh práce MeSH
- software * MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Single cells are basic physiological and biological units that can function individually as well as in groups in tissues and organs. It is central to identify, characterize and profile single cells at molecular level to be able to distinguish different kinds, to understand their functions and determine how they interact with each other. During the last decade several technologies for single-cell profiling have been developed and used in various applications, revealing many novel findings. Quantitative PCR (qPCR) is one of the most developed methods for single-cell profiling that can be used to interrogate several analytes, including DNA, RNA and protein. Single-cell qPCR has the potential to become routine methodology but the technique is still challenging, as it involves several experimental steps and few molecules are handled. Here, we discuss technical aspects and provide recommendation for single-cell qPCR analysis. The workflow includes experimental design, sample preparation, single-cell collection, direct lysis, reverse transcription, preamplification, qPCR and data analysis. Detailed reporting and sharing of experimental details and data will promote further development and make validation studies possible. Efforts aiming to standardize single-cell qPCR open up means to move single-cell analysis from specialized research settings to standard research laboratories.
Background: The Human Cell Differentiation Molecules (HCDM) organizes Human Leukocyte Differentiation Antigen (HLDA) workshops to test and name clusters of antibodies that react with a specific antigen. These cluster of differentiation (CD) markers have provided the scientific community with validated antibody clones, consistent naming of targets and reproducible identification of leukocyte subsets. Still, quantitative CD marker expression profiles and benchmarking of reagents at the single-cell level are currently lacking. Objective: To develop a flow cytometric procedure for quantitative expression profiling of surface antigens on blood leukocyte subsets that is standardized across multiple research laboratories. Methods: A high content framework to evaluate the titration and reactivity of Phycoerythrin (PE)-conjugated monoclonal antibodies (mAbs) was created. Two flow cytometry panels were designed: an innate cell tube for granulocytes, dendritic cells, monocytes, NK cells and innate lymphoid cells (12-color) and an adaptive lymphocyte tube for naive and memory B and T cells, including TCRγδ+, regulatory-T and follicular helper T cells (11-color). The potential of these 2 panels was demonstrated via expression profiling of selected CD markers detected by PE-conjugated antibodies and evaluated using 561 nm excitation. Results: Using automated data annotation and dried backbone reagents, we reached a robust workflow amenable to processing hundreds of measurements in each experiment in a 96-well plate format. The immunophenotyping panels enabled discrimination of 27 leukocyte subsets and quantitative detection of the expression of PE-conjugated CD markers of interest that could quantify protein expression above 400 units of antibody binding capacity. Expression profiling of 4 selected CD markers (CD11b, CD31, CD38, CD40) showed high reproducibility across centers, as well as the capacity to benchmark unique clones directed toward the same CD3 antigen. Conclusion: We optimized a procedure for quantitative expression profiling of surface antigens on blood leukocyte subsets. The workflow, bioinformatics pipeline and optimized flow panels enable the following: 1) mapping the expression patterns of HLDA-approved mAb clones to CD markers; 2) benchmarking new antibody clones to established CD markers; 3) defining new clusters of differentiation in future HLDA workshops.
- MeSH
- antigeny povrchové * metabolismus MeSH
- buňky NK metabolismus MeSH
- CD antigeny metabolismus MeSH
- leukocyty MeSH
- lidé MeSH
- monoklonální protilátky MeSH
- přirozená imunita * MeSH
- průběh práce MeSH
- průtoková cytometrie metody MeSH
- referenční standardy MeSH
- reprodukovatelnost výsledků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The interest to analyze single and few cell samples is rapidly increasing. Numerous extraction protocols to purify nucleic acids are available, but most of them compromise severely on yield to remove contaminants and are therefore not suitable for the analysis of samples containing small numbers of transcripts only. Here, we evaluate 17 direct cell lysis protocols for transcript yield and compatibility with downstream reverse transcription quantitative real-time PCR. Four endogenously expressed genes are assayed together with RNA and DNA spikes in the samples. We found bovine serum albumin (BSA) to be the best lysis agent, resulting in efficient cell lysis, high RNA stability, and enhanced reverse transcription efficiency. Furthermore, we found direct cell lysis with BSA superior to standard column based extraction methods, when analyzing from 1 up to 512 mammalian cells. In conclusion, direct cell lysis protocols based on BSA can be applied with most cell collection methods and are compatible with most analytical workflows to analyze single-cells as well as samples composed of small numbers of cells.
- Publikační typ
- časopisecké články MeSH
Flow cytometry has revolutionized the field of molecular immunology, enabling the monitoring and characterization of immune events at the single-cell level. Here, we describe a flow cytometry-based workflow to quantify the activation of specific immune cell subsets in mice in response to a molecular intervention. Compared to laborious long-term disease models, this technique allows for relatively rapid evaluation of candidate therapeutics designed to elicit a targeted immune response. This approach has the range to address both disease applications in which an immunostimulatory effect would be desired (e.g., cancer, infectious disease) or those in which an immunosuppressive effect would be desired (e.g., autoimmune disorders, transplantation medicine). Overall, our technique presents a powerful and accessible strategy for preliminary in vivo assessment of potential immunotherapeutics.
- MeSH
- analýza jednotlivých buněk metody MeSH
- CD4-pozitivní T-lymfocyty transplantace MeSH
- CD8-pozitivní T-lymfocyty transplantace MeSH
- imunofenotypizace MeSH
- myši transgenní MeSH
- myši MeSH
- nádory imunologie terapie MeSH
- ovalbumin aplikace a dávkování imunologie MeSH
- převzatá imunita MeSH
- průběh práce MeSH
- průtoková cytometrie MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
The advances in electron cryo-microscopy have enabled high-resolution structural studies of vitrified macromolecular complexes in situ by cryo-electron tomography (cryo-ET). Since utilization of cryo-ET is generally limited to the specimens with thickness < 500 nm, a complex sample preparation protocol to study larger samples such as single eukaryotic cells by cryo-ET was developed and optimized over the last decade. The workflow is based on the preparation of a thin cellular lamella by cryo-focused ion beam milling (cryo-FIBM) from the vitrified cells. The sample preparation protocol is a multi-step process which includes utilization of several high-end instruments and comprises sample manipulation prone to sample deterioration. Here, we present a workflow for preparation of three different model specimens that was optimized to provide high-quality lamellae for cryo-ET or electron diffraction tomography with high reproducibility. Preparation of lamellae from large adherent mammalian cells, small suspension eukaryotic cell line, and protein crystals of intermediate size is described which represents examples of the most frequently studied samples used for cryo-FIBM in life sciences.
- MeSH
- buňky ultrastruktura MeSH
- elektronová kryomikroskopie metody MeSH
- ionty MeSH
- makromolekulární látky ultrastruktura MeSH
- molekulární biologie metody MeSH
- odběr biologického vzorku metody MeSH
- proteiny ultrastruktura MeSH
- průběh práce MeSH
- reprodukovatelnost výsledků MeSH
- Saccharomyces cerevisiae ultrastruktura MeSH
- tomografie elektronová metody MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Analyzing rare DNA and RNA molecules in limited sample sizes, such as liquid biopsies and single cells, often requires preamplification, which makes downstream analyses particularly sensitive to polymerase chain reaction (PCR) generated contamination. Herein, we assessed the feasibility of performing Cod uracil-DNA N-glycosylase (Cod UNG) treatment in combination with targeted preamplification, using deoxyuridine triphosphate (dUTP) to eliminate carry-over DNA. Cod UNG can be completely and irreversibly heat inactivated, a prerequisite in preamplification methods, where any loss of amplicons is detrimental to subsequent quantification. Using 96 target assays and quantitative real-time PCR, we show that replacement of deoxythymidine triphosphate (dTTP) with dUTP in the preamplification reaction mix results in comparable dynamic range, reproducibility, and sensitivity. Moreover, Cod UNG essentially removes all uracil-containing template of most assays, regardless of initial concentration, without affecting downstream analyses. Finally, we demonstrate that the use of Cod UNG and dUTP in targeted preamplification can easily be included in the workflow for single-cell gene expression profiling. In summary, Cod UNG treatment in combination with targeted preamplification using dUTP provides a simple and efficient solution to eliminate carry-over contamination and the generation of false positives and inaccurate quantification.
- MeSH
- analýza jednotlivých buněk MeSH
- deoxyuracilnukleotidy metabolismus MeSH
- Gadus morhua metabolismus MeSH
- kontaminace DNA * MeSH
- reprodukovatelnost výsledků MeSH
- stanovení celkové genové exprese MeSH
- uracil-DNA-glykosidasa metabolismus MeSH
- uracil metabolismus MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
