TY  - JOUR
AB  - This protocol provides comprehensive guidance for researchers, including those with limited electron microscopy (EM) experience, on the effective use of genetically encoded, multichannel EMcapsulin reporters in both fluorescence microscopy and EM workflows. EMcapsulins are a set of modular gene reporters that manifest as distinct shapes in EM when expressed in cell culture or model organisms. This protocol encompasses detailed instructions for labeling cells or proteins of interest with fluorescent EMcapsulins, along with biochemical quality control measures. Researchers may also adapt the protocol to develop custom EMcapsulin constructs tailored to their specific experimental requirements.
AU  - Berezin, O.
AU  - Piovesan, A.
AU  - Graf, R.
AU  - Samara, E.*
AU  - Sigmund, F.
AU  - Westmeyer, G.G.
C1  - 76090
C2  - 58407
TI  - Multiplexed genetic tags for electron and fluorescence microscopy.
JO  - Nat. Protoc.
PY  - 2025
SN  - 1754-2189
ER  - 

TY  - JOUR
AB  - Spheroids are reaggregated multicellular three-dimensional structures generated from cells or cell cultures of healthy as well as pathological tissue. Basic and translational spheroid application across academia and industry have led to the development of multiple setups and analysis methods, which mostly lack the modularity to maximally phenotype spheroids. Here we present the self-assembly of single-cell suspensions into spheroids by the liquid overlay method, followed by a modular framework for a multifaceted phenotyping of spheroids. Cell seeding, supernatant handling and compound administration are elaborated by both manual and automated procedures. The phenotyping modules contain a suite of orthogonal assays to analyze spheroids longitudinally and/or at an endpoint. Longitudinal analyses include morphometry with or without spheroid or cell state specific information and supernatant evaluation (nutrient consumption and metabolite/cytokine production). Spheroids can also be used as a starting point to monitor single and collective cell migration and invasion. At an endpoint, spheroids are lysed, fixed or dissociated into single cells. Endpoint analyses allow the investigation of molecular content, single-cell composition and state and architecture with spatial cell and subcellular specific information. Each module addresses time requirements and quality control indicators to support reproducibility. The presented complementary techniques can be readily adopted by researchers experienced in cell culture and basic molecular biology. We anticipate that this modular protocol will advance the application of three-dimensional biology by providing scalable and complementary methods.
AU  - Blondeel, E.*
AU  - Ernst, S.W.*
AU  - De Vuyst, F.*
AU  - Diosdi, A.*
AU  - Pinheiro, C.*
AU  - Estêvão, D.*
AU  - Rappu, P.*
AU  - Boiy, R.*
AU  - Dedeyne, S.*
AU  - Craciun, L.*
AU  - Goossens, V.*
AU  - Dehairs, J.*
AU  - Cruz, T.*
AU  - Audenaert, D.*
AU  - Ceelen, W.*
AU  - Linnebacher, M.*
AU  - Boterberg, T.*
AU  - Vandesompele, J.*
AU  - Mestdagh, P.*
AU  - Swinnen, J.*
AU  - Heino, J.*
AU  - Horvath, P.
AU  - Oliveira, M.J.*
AU  - Hendrix, A.*
AU  - De Metter, P.*
AU  - De Wever, O.*
C1  - 74030
C2  - 57299
CY  - Heidelberger Platz 3, Berlin, 14197, Germany
SP  - 2899-2941
TI  - Sequential orthogonal assays for longitudinal and endpoint characterization of three-dimensional spheroids.
JO  - Nat. Protoc.
VL  - 20
IS  - 10
PB  - Nature Portfolio
PY  - 2025
SN  - 1754-2189
ER  - 

TY  - JOUR
AB  - Correction to: Nature Protocolshttps://doi.org/10.1038/s41596-025-01150-y, published online 8 April 2025. In the version of the article initially published, Pieter Demetter’s surname appeared incorrectly (as De Metter) and has now been amended in the HTML and PDF versions of the article.
AU  - Blondeel, E.*
AU  - Ernst, S.W.*
AU  - De Vuyst, F.*
AU  - Diosdi, A.*
AU  - Pinheiro, C.*
AU  - Estêvão, D.*
AU  - Rappu, P.*
AU  - Boiy, R.*
AU  - Dedeyne, S.*
AU  - Craciun, L.*
AU  - Goossens, V.*
AU  - Dehairs, J.*
AU  - Cruz, T.*
AU  - Audenaert, D.*
AU  - Ceelen, W.*
AU  - Linnebacher, M.*
AU  - Boterberg, T.*
AU  - Vandesompele, J.*
AU  - Mestdagh, P.*
AU  - Swinnen, J.*
AU  - Heino, J.*
AU  - Horvath, P.
AU  - Oliveira, M.J.*
AU  - Hendrix, A.*
AU  - Demetter, P.*
AU  - De Wever, O.*
C1  - 74893
C2  - 57652
TI  - Author Correction: Sequential orthogonal assays for longitudinal and endpoint characterization of three-dimensional spheroids.
JO  - Nat. Protoc.
PY  - 2025
SN  - 1754-2189
ER  - 

TY  - JOUR
AB  - Hematoxylin- and eosin-stained whole-slide images (WSIs) are the foundation of diagnosis of cancer. In recent years, development of deep learning-based methods in computational pathology has enabled the prediction of biomarkers directly from WSIs. However, accurately linking tissue phenotype to biomarkers at scale remains a crucial challenge for democratizing complex biomarkers in precision oncology. This protocol describes a practical workflow for solid tumor associative modeling in pathology (STAMP), enabling prediction of biomarkers directly from WSIs by using deep learning. The STAMP workflow is biomarker agnostic and allows for genetic and clinicopathologic tabular data to be included as an additional input, together with histopathology images. The protocol consists of five main stages that have been successfully applied to various research problems: formal problem definition, data preprocessing, modeling, evaluation and clinical translation. The STAMP workflow differentiates itself through its focus on serving as a collaborative framework that can be used by clinicians and engineers alike for setting up research projects in the field of computational pathology. As an example task, we applied STAMP to the prediction of microsatellite instability (MSI) status in colorectal cancer, showing accurate performance for the identification of tumors high in MSI. Moreover, we provide an open-source code base, which has been deployed at several hospitals across the globe to set up computational pathology workflows. The STAMP workflow requires one workday of hands-on computational execution and basic command line knowledge.
AU  - El Nahhas, O.S.M.*
AU  - van Treeck, M.*
AU  - Wölflein, G.*
AU  - Unger, M.*
AU  - Ligero, M.*
AU  - Lenz, T.*
AU  - Wagner, S.
AU  - Hewitt, K.J.*
AU  - Khader, F.*
AU  - Foersch, S.*
AU  - Truhn, D.*
AU  - Kather, J.N.*
C1  - 71733
C2  - 56391
CY  - Heidelberger Platz 3, Berlin, 14197, Germany
TI  - From whole-slide image to biomarker prediction: End-to-end weakly supervised deep learning in computational pathology.
JO  - Nat. Protoc.
PB  - Nature Portfolio
PY  - 2024
SN  - 1754-2189
ER  - 

TY  - JOUR
AB  - Volume electron microscopy is the method of choice for the in situ interrogation of cellular ultrastructure at the nanometer scale, and with the increase in large raw image datasets generated, improving computational strategies for image segmentation and spatial analysis is necessary. Here we describe a practical and annotation-efficient pipeline for organelle-specific segmentation, spatial analysis and visualization of large volume electron microscopy datasets using freely available, user-friendly software tools that can be run on a single standard workstation. The procedures are aimed at researchers in the life sciences with modest computational expertise, who use volume electron microscopy and need to generate three-dimensional (3D) segmentation labels for different types of cell organelles while minimizing manual annotation efforts, to analyze the spatial interactions between organelle instances and to visualize the 3D segmentation results. We provide detailed guidelines for choosing well-suited segmentation tools for specific cell organelles, and to bridge compatibility issues between freely available open-source tools, we distribute the critical steps as easily installable Album solutions for deep learning segmentation, spatial analysis and 3D rendering. Our detailed description can serve as a reference for similar projects requiring particular strategies for single- or multiple-organelle analysis, which can be achieved with computational resources commonly available to single-user setups.
AU  - Müller, A.
AU  - Schmidt, D.*
AU  - Albrecht, J.P.*
AU  - Rieckert, L.*
AU  - Otto, M.*
AU  - Galicia Garcia, L.E.
AU  - Fabig, G.*
AU  - Solimena, M.
AU  - Weigert, M.*
C1  - 70100
C2  - 55420
CY  - Heidelberger Platz 3, Berlin, 14197, Germany
TI  - Modular segmentation, spatial analysis and visualization of volume electron microscopy datasets.
JO  - Nat. Protoc.
PB  - Nature Portfolio
PY  - 2024
SN  - 1754-2189
ER  - 

TY  - JOUR
AB  - Homeostatic and pathological phenomena often affect multiple organs across the whole organism. Tissue clearing methods, together with recent advances in microscopy, have made holistic examinations of biological samples feasible. Here, we report the detailed protocol for nanobody(VHH)-boosted 3D imaging of solvent-cleared organs (vDISCO), a pressure-driven, nanobody-based whole-body immunolabeling and clearing method that renders whole mice transparent in 3 weeks, consistently enhancing the signal of fluorescent proteins, stabilizing them for years. This allows the reliable detection and quantification of fluorescent signal in intact rodents enabling the analysis of an entire body at cellular resolution. Here, we show the high versatility of vDISCO applied to boost the fluorescence signal of genetically expressed reporters and clear multiple dissected organs and tissues, as well as how to image processed samples using multiple fluorescence microscopy systems. The entire protocol is accessible to laboratories with limited expertise in tissue clearing. In addition to its applications in obtaining a whole-mouse neuronal projection map, detecting single-cell metastases in whole mice and identifying previously undescribed anatomical structures, we further show the visualization of the entire mouse lymphatic system, the application for virus tracing and the visualization of all pericytes in the brain. Taken together, our vDISCO pipeline allows systematic and comprehensive studies of cellular phenomena and connectivity in whole bodies.
AU  - Cai, R.
AU  - Kolabas, Z.I.
AU  - Pan, C.
AU  - Mai, H.
AU  - Zhao, S.
AU  - Kaltenecker, D.
AU  - Voigt, F.F.*
AU  - Molbay, M.
AU  - Ohn, T.-L.
AU  - Vincke, C.*
AU  - Todorov, M.I.
AU  - Helmchen, F.*
AU  - Van Ginderachter, J.A.*
AU  - Ertürk, A.
C1  - 67356
C2  - 54190
SP  - 1197-1242
TI  - Whole-mouse clearing and imaging at the cellular level with vDISCO.
JO  - Nat. Protoc.
VL  - 18
IS  - 4
PY  - 2023
SN  - 1754-2189
ER  - 

TY  - JOUR
AB  - The comet assay is a versatile method to detect nuclear DNA damage in individual eukaryotic cells, from yeast to human. The types of damage detected encompass DNA strand breaks and alkali-labile sites (e.g., apurinic/apyrimidinic sites), alkylated and oxidized nucleobases, DNA-DNA crosslinks, UV-induced cyclobutane pyrimidine dimers and some chemically induced DNA adducts. Depending on the specimen type, there are important modifications to the comet assay protocol to avoid the formation of additional DNA damage during the processing of samples and to ensure sufficient sensitivity to detect differences in damage levels between sample groups. Various applications of the comet assay have been validated by research groups in academia, industry and regulatory agencies, and its strengths are highlighted by the adoption of the comet assay as an in vivo test for genotoxicity in animal organs by the Organisation for Economic Co-operation and Development. The present document includes a series of consensus protocols that describe the application of the comet assay to a wide variety of cell types, species and types of DNA damage, thereby demonstrating its versatility.
AU  - Collins, A.*
AU  - Møller, P.*
AU  - Gajski, G.*
AU  - Vodenková, S.*
AU  - Abdulwahed, A.*
AU  - Anderson, D.*
AU  - Bankoglu, E.E.*
AU  - Bonassi, S.*
AU  - Boutet-Robinet, E.*
AU  - Brunborg, G.*
AU  - Chao, C.*
AU  - Cooke, M.S.*
AU  - Costa, C.*
AU  - Costa, S.*
AU  - Dhawan, A.*
AU  - de Lapuente, J.*
AU  - Bo', C.D.*
AU  - Dubus, J.*
AU  - Dusinska, M.*
AU  - Duthie, S.J.*
AU  - Yamani, N.E.*
AU  - Engelward, B.*
AU  - Gaivão, I.*
AU  - Giovannelli, L.*
AU  - Godschalk, R.*
AU  - Guilherme, S.*
AU  - Gutzkow, K.B.*
AU  - Habas, K.*
AU  - Hernandez, A.*
AU  - Herrero, O.*
AU  - Isidori, M.*
AU  - Jha, A.N.*
AU  - Knasmüller, S.*
AU  - Kooter, I.M.*
AU  - Koppen, G.*
AU  - Kruszewski, M.*
AU  - Ladeira, C.*
AU  - Laffon, B.*
AU  - Larramendy, M.*
AU  - Hégarat, L.L.*
AU  - Lewies, A.*
AU  - Lewinska, A.*
AU  - Liwszyc, G.E.*
AU  - de Cerain, A.L.*
AU  - Manjanatha, M.*
AU  - Marcos, R.*
AU  - Milić, M.*
AU  - de Andrade, V.M.*
AU  - Moretti, M.*
AU  - Muruzabal, D.*
AU  - Novak, M.*
AU  - Oliveira, R.*
AU  - Olsen, A.K.*
AU  - Owiti, N.*
AU  - Pacheco, M.*
AU  - Pandey, A.K.*
AU  - Pfuhler, S.*
AU  - Pourrut, B.*
AU  - Reisinger, K.*
AU  - Rojas, E.*
AU  - Rundén-Pran, E.*
AU  - Sanz-Serrano, J.*
AU  - Shaposhnikov, S.*
AU  - Sipinen, V.*
AU  - Smeets, K.*
AU  - Stopper, H.*
AU  - Teixeira, J.P.*
AU  - Valdiglesias, V.*
AU  - Valverde, M.*
AU  - van Acker, F.*
AU  - van Schooten, F.J.*
AU  - Vasquez, M.*
AU  - Wentzel, J.F.*
AU  - Wnuk, M.*
AU  - Wouters, A.*
AU  - Žegura, B.*
AU  - Zikmund, T.
AU  - Langie, S.A.S.*
AU  - Azqueta, A.*
C1  - 67341
C2  - 54179
SP  - 929-989
TI  - Measuring DNA modifications with the comet assay: A compendium of protocols.
JO  - Nat. Protoc.
VL  - 18
IS  - 3
PY  - 2023
SN  - 1754-2189
ER  - 

TY  - JOUR
AB  - Connective tissues are essential building blocks for organ development, repair and regeneration. However, we are at the early stages of understanding connective tissue dynamics. Here, we detail a method that enables in vivo fate mapping of organ extracellular matrix (ECM) by taking advantage of a crosslinking chemical reaction between amine groups and N-hydroxysuccinimide esters. This methodology enables robust labeling of ECM proteins, which complement previous affinity-based single-protein methods. This protocol is intended for entry-level scientists and the labeling step takes between 5 and 10 min. ECM 'tagging' with fluorophores using N-hydroxysuccinimide esters enables visualization of ECM spatial modifications and is particularly useful to study connective tissue dynamics in organ fibrosis, tumor stroma formation, wound healing and regeneration. This in vivo chemical fate mapping methodology is highly versatile, regardless of the tissue/organ system, and complements cellular fate-mapping techniques. Furthermore, as the basic chemistry of proteins is highly conserved between species, this method is also suitable for cross-species comparative studies of ECM dynamics.
AU  - Fischer, A.
AU  - Correa-Gallegos, D.
AU  - Wannemacher, J.
AU  - Christ, S.
AU  - Machens, H.G.*
AU  - Rinkevich, Y.
C1  - 68012
C2  - 54490
CY  - Heidelberger Platz 3, Berlin, 14197, Germany
SP  - 2876-2890
TI  - In vivo fluorescent labeling and tracking of extracellular matrix.
JO  - Nat. Protoc.
VL  - 18
IS  - 10
PB  - Nature Portfolio
PY  - 2023
SN  - 1754-2189
ER  - 

TY  - JOUR
AB  - The human gut microbiome is a key contributor to health, and its perturbations are linked to many diseases. Small-molecule xenobiotics such as drugs, chemical pollutants and food additives can alter the microbiota composition and are now recognized as one of the main factors underlying microbiome diversity. Mapping the effects of such compounds on the gut microbiome is challenging because of the complexity of the community, anaerobic growth requirements of individual species and the large number of interactions that need to be quantitatively assessed. High-throughput screening setups offer a promising solution for probing the direct inhibitory effects of hundreds of xenobiotics on tens of anaerobic gut bacteria. When automated, such assays enable the cost-effective investigation of a wide range of compound-microbe combinations. We have developed an experimental setup and protocol that enables testing of up to 5,000 compounds on a target gut species under strict anaerobic conditions within 5 d. In addition, with minor modifications to the protocol, drug effects can be tested on microbial communities either assembled from isolates or obtained from stool samples. Experience in working in an anaerobic chamber, especially in performing delicate work with thick chamber gloves, is required for implementing this protocol. We anticipate that this protocol will accelerate the study of interactions between small molecules and the gut microbiome and provide a deeper understanding of this microbial ecosystem, which is intimately intertwined with human health.
AU  - Müller, P.*
AU  - de la Cuesta-Zuluaga, J.*
AU  - Kuhn, M.*
AU  - Baghai Arassi, M.*
AU  - Treis, T.
AU  - Blasche, S.*
AU  - Zimmermann, M.*
AU  - Bork, P.*
AU  - Patil, K.R.*
AU  - Typas, A.*
AU  - Garcia-Santamarina, S.*
AU  - Maier, L.A.*
C1  - 68975
C2  - 53793
CY  - Heidelberger Platz 3, Berlin, 14197, Germany
SP  - 38
TI  - High-throughput anaerobic screening for identifying compounds acting against gut bacteria in monocultures or communities.
JO  - Nat. Protoc.
PB  - Nature Portfolio
PY  - 2023
SN  - 1754-2189
ER  - 

TY  - JOUR
AB  - Mitochondria are key bioenergetic organelles involved in many biosynthetic and signaling pathways. However, their differential contribution to specific functions of cells within complex tissues is difficult to dissect with current methods. The present protocol addresses this need by enabling the ex vivo immunocapture of cell-type-specific mitochondria directly from their tissue context through a MitoTag reporter mouse. While other available methods were developed for bulk mitochondria isolation or more abundant cell-type-specific mitochondria, this protocol was optimized for the selective isolation of functional mitochondria from medium-to-low-abundant cell types in a heterogeneous tissue, such as the central nervous system. The protocol has three major parts: First, mitochondria of a cell type of interest are tagged via an outer mitochondrial membrane eGFP by crossing MitoTag mice to a cell-type-specific Cre-driver line or by delivery of viral vectors for Cre expression. Second, homogenates are prepared from relevant tissues by nitrogen cavitation, from which tagged organelles are immunocaptured using magnetic microbeads. Third, immunocaptured mitochondria are used for downstream assays, e.g., to probe respiratory capacity or calcium handling, revealing cell-type-specific mitochondrial diversity in molecular composition and function. The MitoTag approach enables the identification of marker proteins to label cell-type-specific organelle populations in situ, elucidates cell-type-enriched mitochondrial metabolic and signaling pathways, and reveals functional mitochondrial diversity between adjacent cell types in complex tissues, such as the brain. Apart from establishing the mouse colony (6–8 weeks without import), the immunocapture protocol takes 2 h and functional assays require 1–2 h.
AU  - Prudente de Mello, N.
AU  - Fecher, C.*
AU  - Pastor, A.M.*
AU  - Perocchi, F.
AU  - Misgeld, T.*
C1  - 68503
C2  - 54682
CY  - Heidelberger Platz 3, Berlin, 14197, Germany
SP  - 2181-2220
TI  - Ex vivo immunocapture and functional characterization of cell-type-specific mitochondria using MitoTag mice.
JO  - Nat. Protoc.
VL  - 18
IS  - 7
PB  - Nature Portfolio
PY  - 2023
SN  - 1754-2189
ER  - 

TY  - JOUR
AB  - Advances in tissue labeling and clearing methods include improvement of tissue transparency, better preservation of fluorescence signal, compatibility with immunostaining and large sample volumes. However, as existing methods share the common limitation that they can only be applied to human tissue slices, rendering intact human organs transparent remains a challenge. Here, we describe experimental details of the small-micelle-mediated human organ efficient clearing and labeling (SHANEL) pipeline, which can be applied for cellular mapping of intact human organs. We have successfully cleared multiple human organs, including kidney, pancreas, heart, lung, spleen and brain, as well as hard tissue like skull. We also describe an advanced volumetric imaging system using a commercial light-sheet fluorescence microscope that can accommodate most human organs and a pipeline for whole-organ imaging and visualization. The complete experimental process of labeling and clearing whole human organs takes months and the analysis process takes several weeks, depending on the organ types and sizes.
AU  - Mai, H.
AU  - Rong, Z.
AU  - Zhao, S.
AU  - Cai, R.
AU  - Steinke, H.*
AU  - Bechmann, I.*
AU  - Ertürk, A.
C1  - 65764
C2  - 52905
SP  - 2188-2215
TI  - Scalable tissue labeling and clearing of intact human organs.
JO  - Nat. Protoc.
VL  - 17
IS  - 10
PY  - 2022
SN  - 1754-2189
ER  - 

TY  - JOUR
AB  - The clonogenic assay measures the capacity of single cells to form colonies in vitro. It is widely used to identify and quantify self-renewing mammalian cells derived from in vitro cultures as well as from ex vivo tissue preparations of different origins. Varying research questions and the heterogeneous growth requirements of individual cell model systems led to the development of several assay principles and formats that differ with regard to their conceptual setup, 2D or 3D culture conditions, optional cytotoxic treatments and subsequent mathematical analysis. The protocol presented here is based on the initial clonogenic assay protocol as developed by Puck and Marcus more than 60 years ago. It updates and extends the 2006 Nature Protocols article by Franken et al. It discusses different strategies and principles to analyze clonogenic growth in vitro and presents the clonogenic assay in a modular protocol framework enabling a diversity of formats and measures to optimize determination of clonogenic growth parameters. We put particular focus on the phenomenon of cellular cooperation and consideration of how this can affect the mathematical analysis of survival data. This protocol is applicable to any mammalian cell model system from which single-cell suspensions can be prepared and which contains at least a small fraction of cells with self-renewing capacity in vitro. Depending on the cell system used, the entire procedure takes ~2–10 weeks, with a total hands-on time of <20 h per biological replicate.
AU  - Brix, N.*
AU  - Samaga, D.
AU  - Belka, C.
AU  - Zitzelsberger, H.
AU  - Lauber, K.
C1  - 63374
C2  - 51306
CY  - Heidelberger Platz 3, Berlin, 14197, Germany
SP  - 4963-4991
TI  - Analysis of clonogenic growth in vitro.
JO  - Nat. Protoc.
VL  - 16
IS  - 11
PB  - Nature Portfolio
PY  - 2021
SN  - 1754-2189
ER  - 

TY  - JOUR
AB  - The recent CRISPR revolution has provided researchers with powerful tools to perform genome editing in a variety of organisms. However, recent reports indicate widespread occurrence of unintended CRISPR-induced on-target effects (OnTEs) at the edited site in mice and human induced pluripotent stem cells (iPSCs) that escape standard quality controls. By altering gene expression of targeted or neighbouring genes, OnTEs can severely affect phenotypes of CRISPR-edited cells and organisms and thus lead to data misinterpretation, which can undermine the reliability of CRISPR-based studies. Here we describe a broadly applicable framework for detecting OnTEs in genome-edited cells and organisms after non-homologous end joining-mediated and homology-directed repair-mediated editing. Our protocol enables identification of OnTEs such as large deletions, large insertions, rearrangements or loss of heterozygosity (LOH). This is achieved by subjecting genomic DNA first to quantitative genotyping PCR (qgPCR), which determines the number of intact alleles at the target site using the same PCR amplicon that has been optimized for genotyping. This combination of genotyping and quantitation makes it possible to exclude clones with monoallelic OnTEs and hemizygous editing, which are often mischaracterized as correctly edited in standard Sanger sequencing. Second, occurrence of LOH around the edited locus is detected by genotyping neighbouring single-nucleotide polymorphisms (SNPs), using either a Sanger sequencing-based method or SNP microarrays. All steps are optimized to maximize simplicity and minimize cost to promote wide dissemination and applicability across the field. The entire protocol from genomic DNA extraction to OnTE exclusion can be performed in 6–9 d.
AU  - Weisheit, I.*
AU  - Kroeger, J.A.*
AU  - Malik, R.*
AU  - Wefers, B.
AU  - Lichtner, P.
AU  - Wurst, W.
AU  - Dichgans, M.*
AU  - Paquet, D.*
C1  - 61403
C2  - 50212
CY  - Heidelberger Platz 3, Berlin, 14197, Germany
SP  - 1714-1739
TI  - Simple and reliable detection of CRISPR-induced on-target effects by qgPCR and SNP genotyping.
JO  - Nat. Protoc.
VL  - 16
IS  - 3
PB  - Nature Research
PY  - 2021
SN  - 1754-2189
ER  - 

TY  - JOUR
AB  - RNA sequencing (RNA-seq) has emerged as a powerful approach to discover disease-causing gene regulatory defects in individuals affected by genetically undiagnosed rare disorders. Pioneering studies have shown that RNA-seq could increase the diagnosis rates over DNA sequencing alone by 8-36%, depending on the disease entity and tissue probed. To accelerate adoption of RNA-seq by human genetics centers, detailed analysis protocols are now needed. We present a step-by-step protocol that details how to robustly detect aberrant expression levels, aberrant splicing and mono-allelic expression in RNA-seq data using dedicated statistical methods. We describe how to generate and assess quality control plots and interpret the analysis results. The protocol is based on the detection of RNA outliers pipeline (DROP), a modular computational workflow that integrates all the analysis steps, can leverage parallel computing infrastructures and generates browsable web page reports.
AU  - Yépez, V.A.*
AU  - Mertes, C.*
AU  - Müller, M.F.*
AU  - Wachutka, L.*
AU  - Frésard, L.*
AU  - Gusic, M.
AU  - Scheller, I.F.*
AU  - Goldberg, P.F.*
AU  - Prokisch, H.
AU  - Gagneur, J.
C1  - 61091
C2  - 49682
CY  - Heidelberger Platz 3, Berlin, 14197, Germany
SP  - 1276–1296
TI  - Detection of aberrant gene expression events in RNA sequencing data.
JO  - Nat. Protoc.
VL  - 16
PB  - Nature Research
PY  - 2021
SN  - 1754-2189
ER  - 

TY  - JOUR
AB  - Suitable membrane mimetics are crucial to the performance of structural and functional studies of membrane proteins. Phospholipid nanodiscs (formed when a membrane scaffold protein encircles a small portion of a lipid bilayer) have native-like membrane properties. These have been used for a variety of functional studies, but structural studies by high-resolution solution-state NMR spectroscopy of membrane proteins in commonly used nanodiscs of 10-nm diameter were limited by the high molecular weight of these particles, which caused unfavorably large NMR line widths. We have recently constructed truncated versions of the membrane scaffold protein, allowing the preparation of a range of stepwise-smaller nanodiscs (6- to 8-nm diameter) to overcome this limitation. Here, we present a protocol on the assembly of phospholipid nanodiscs of various sizes for structural studies of membrane proteins with solution-state NMR spectroscopy. We describe specific isotope-labeling schemes required for working with large membrane protein systems in nanodiscs, and provide guidelines on the setup of NMR non-uniform sampling (NUS) data acquisition and high-resolution NMR spectra reconstruction. We discuss critical points and pitfalls relating to optimization of nanodiscs for NMR spectroscopy and outline a strategy for the high-resolution structure determination and positioning of isotope-labeled membrane proteins in nanodiscs using nuclear Overhauser enhancement spectroscopy (NOESY) spectroscopy, residual dipolar couplings (RDCs) and paramagnetic relaxation enhancements (PREs). Depending on the target protein of interest, nanodisc assembly and purification can be achieved within 12-24 h. Although the focus of this protocol is on protein NMR, these nanodiscs can also be used for (cryo-) electron microscopy (EM) and small-angle X-ray and neutron-scattering studies.
AU  - Hagn, F.
AU  - Nasr, M.L.*
AU  - Wagner, G.*
C1  - 52651
C2  - 44096
CY  - London
SP  - 79-98
TI  - Assembly of phospholipid nanodiscs of controlled size for structural studies of membrane proteins by NMR.
JO  - Nat. Protoc.
VL  - 13
IS  - 1
PB  - Nature Publishing Group
PY  - 2018
SN  - 1754-2189
ER  - 

TY  - JOUR
AB  - Adult neural stem cells (aNSCs) in zebrafish produce mature neurons throughout their entire life span in both the intact and regenerating brain. An understanding of the behavior of aNSCs in their intact niche and during regeneration in vivo should facilitate the identification of the molecular mechanisms controlling regeneration-specific cellular events. A greater understanding of the process in regeneration-competent species may enable regeneration to be achieved in regeneration-incompetent species, including humans. Here we describe a protocol for labeling and repetitive imaging of aNSCs in vivo. We label single aNSCs, allowing nonambiguous re-identification of single cells in repetitive imaging sessions using electroporation of a red-reporter plasmid in Tg(gfap:GFP)mi2001 transgenic fish expressing GFP in aNSCs. We image using two-photon microscopy through the thinned skull of anesthetized and immobilized fish. Our protocol allows imaging every 2 d for a period of up to 1 month. This methodology allowed the visualization of aNSC behavior in vivo in their natural niche, in contrast to previously available technologies, which rely on the imaging of either dissociated cells or tissue slices. We used this protocol to follow the mode of aNSC division, fate changes and cell death in both the intact and injured zebrafish telencephalon. This experimental setup can be widely used, with minimal prior experience, to assess key factors for processes that modulate aNSC behavior. A typical experiment with data analysis takes up to 1.5 months.
AU  - Barbosa, J.S.
AU  - di Giaimo, R.*
AU  - Götz, M.
AU  - Ninkovic, J.
C1  - 49030
C2  - 41551
CY  - London
SP  - 1360-1370
TI  - Single-cell in vivo imaging of adult neural stem cells in the zebrafish telencephalon.
JO  - Nat. Protoc.
VL  - 11
IS  - 8
PB  - Nature Publishing Group
PY  - 2016
SN  - 1754-2189
ER  - 

TY  - JOUR
AB  - Formalin-fixed and paraffin-embedded (FFPE) tissue specimens are the gold standard for histological examination, and they provide valuable molecular information in tissue-based research. Metabolite assessment from archived tissue samples has not been extensively conducted because of a lack of appropriate protocols and concerns about changes in metabolite content or chemical state due to tissue processing. We present a protocol for the in situ analysis of metabolite content from FFPE samples using a high-mass-resolution matrix-assisted laser desorption/ionization fourier-transform ion cyclotron resonance mass spectrometry imaging (MALDI-FT-ICR-MSI) platform. The method involves FFPE tissue sections that undergo deparaffinization and matrix coating by 9-aminoacridine before MALDI-MSI. Using this platform, we previously detected ∼1,500 m/z species in the mass range m/z 50-1,000 in FFPE samples; the overlap compared with fresh frozen samples is 72% of m/z species, indicating that metabolites are largely conserved in FFPE tissue samples. This protocol can be reproducibly performed on FFPE tissues, including small samples such as tissue microarrays and biopsies. The procedure can be completed in a day, depending on the size of the sample measured and raster size used. Advantages of this approach include easy sample handling, reproducibility, high throughput and the ability to demonstrate molecular spatial distributions in situ. The data acquired with this protocol can be used in research and clinical practice.
AU  - Ly, A.
AU  - Buck, A.
AU  - Balluff, B.*
AU  - Sun, N.
AU  - Gorzolka, K.
AU  - Feuchtinger, A.
AU  - Janssen, K.P.*
AU  - Kuppen, P.J.*
AU  - van de Velde, C.J.*
AU  - Weirich, G.*
AU  - Erlmeier, F.*
AU  - Langer, R.*
AU  - Aubele, M.
AU  - Zitzelsberger, H.
AU  - McDonnell, L.A.*
AU  - Aichler, M.
AU  - Walch, A.K.
C1  - 49083
C2  - 41649
CY  - London
SP  - 1428-1443
TI  - High-mass-resolution MALDI mass spectrometry imaging of metabolites from formalin-fixed paraffin-embedded tissue.
JO  - Nat. Protoc.
VL  - 11
IS  - 8
PB  - Nature Publishing Group
PY  - 2016
SN  - 1754-2189
ER  - 

TY  - JOUR
AB  - Rigorous organization and quality control (QC) are necessary to facilitate successful genome-wide association meta-analyses (GWAMAs) of statistics aggregated across multiple genome-wide association studies. This protocol provides guidelines for (i) organizational aspects of GWAMAs, and for (ii) QC at the study file level, the meta-level across studies and the meta-analysis output level. Real-world examples highlight issues experienced and solutions developed by the GIANT Consortium that has conducted meta-analyses including data from 125 studies comprising more than 330,000 individuals. We provide a general protocol for conducting GWAMAs and carrying out QC to minimize errors and to guarantee maximum use of the data. We also include details for the use of a powerful and flexible software package called EasyQC. Precise timings will be greatly influenced by consortium size. For consortia of comparable size to the GIANT Consortium, this protocol takes a minimum of about 10 months to complete.
AU  - Winkler, T.W.*
AU  - Day, F.R.*
AU  - Croteau-Chonka, D.C.*
AU  - Wood, A.R.*
AU  - Locke, A.E.*
AU  - Mägi, R.*
AU  - Ferreira, T.*
AU  - Fall, T.*
AU  - Graff, M.*
AU  - Justice, A.E.*
AU  - Luan, J.*
AU  - Gustafsson, S.*
AU  - Randall, J.C.*
AU  - Vedantam, S.*
AU  - Workalemahu, T.*
AU  - Kilpeläinen, T.O.*
AU  - Scherag, A.*
AU  - Esko, T.*
AU  - Kutalik, Z.*
AU  - Heid, I.M.
AU  - Loos, R.J.*
AU  - GIANT Consortium (Albrecht, E.
AU  - Müller-Nurasyid, M.
AU  - Grallert, H.
AU  - Peters, A.
AU  - Thorand, B.
AU  - Gieger, C.
AU  - Illig, T.
AU  - Wichmann, H.-E.)
C1  - 31949
C2  - 34884
SP  - 1192-1212
TI  - Quality control and conduct of genome-wide association meta-analyses.
JO  - Nat. Protoc.
VL  - 9
IS  - 5
PY  - 2014
SN  - 1754-2189
ER  - 

TY  - JOUR
AB  - The increasing use of human pluripotent stem cells (hPSCs) as a source of cells for drug discovery, cytotoxicity assessment and disease modeling requires their adaptation to large-scale culture conditions and screening formats. Here, we describe a simple and robust protocol for the adaptation of human embryonic stem cells (hESCs) to high-throughput screening (HTS). This protocol can also be adapted to human induced pluripotent stem cells (hiPSCs) and high-content screening (HCS). We also describe a 7-d assay to identify compounds with an effect on hESC self-renewal and differentiation. This assay can be adapted to a variety of applications. The procedure involves the culture expansion of hESCs, their adaptation to 384-well plates, the addition of small molecules or other factors, and finally data acquisition and processing. In this protocol, the optimal number of hESCs plated in 384-well plates has been adapted to HTS/HCS assays of 7 d.
AU  - Desbordes, S.C.
AU  - Studer, L.*
C1  - 22404
C2  - 30892
SP  - 111-130
TI  - Adapting human pluripotent stem cells to high-throughput and high-content screening.
JO  - Nat. Protoc.
VL  - 8
IS  - 1
PB  - Nature Publishing Group
PY  - 2013
SN  - 1754-2189
ER  - 

TY  - JOUR
AB  - Genetically engineered mice are instrumental for the analysis of mammalian gene function in health and disease. As classical gene targeting, which is performed in embryonic stem (ES) cell cultures and generates chimeric mice, is a time-consuming and labor-intensive procedure, we recently used transcription activator-like (TAL) effector nucleases (TALENs) for mutagenesis of the mouse genome directly in one-cell embryos. Here we describe a stepwise protocol for the generation of knock-in and knockout mice, including the selection of TALEN-binding sites, the design and construction of TALEN coding regions and of mutagenic oligodeoxynucleotides (ODNs) and targeting vectors, mRNA production, embryo microinjection and the identification of modified alleles in founder mutants and their progeny. After a setup time of 2-3 weeks of hands-on work for TALEN construction, investigators can obtain first founder mutants for genes of choice within 7 weeks after embryo microinjections.
AU  - Wefers, B.
AU  - Panda, S.
AU  - Ortiz, O.
AU  - Brandl, C.
AU  - Hensler, S.
AU  - Hansen, J.
AU  - Wurst, W.
AU  - Kühn, R.
C1  - 28613
C2  - 33485
SP  - 2355-2379
TI  - Generation of targeted mouse mutants by embryo microinjection of TALEN mRNA.
JO  - Nat. Protoc.
VL  - 8
IS  - 12
PB  - Nature Publishing
PY  - 2013
SN  - 1754-2189
ER  - 

TY  - JOUR
AB  - Neural stem cells (NSCs) have the remarkable capacity to self-renew and the lifelong ability to generate neurons in the adult mammalian brain. However, the molecular and cellular mechanisms contributing to these behaviors are still not understood. Now that prospective isolation of the NSCs has become feasible, these mechanisms can be studied. Here we describe a protocol for the efficient isolation of adult NSCs, by the application of a dual-labeling strategy on the basis of their glial identity and ciliated nature. The cells are isolated from the lateral ventricular subependymal zone (SEZ) of adult hGFAP-eGFP (human glial fibrillary acidic protein-enhanced green fluorescent protein) transgenic mice by fluorescence-activated cell sorting. Staining against prominin1 (CD133) allows the isolation of the NSCs (hGFAP-eGFP(+)/prominin1(+)), which can be further subdivided by labeling with the fluorescent epidermal growth factor. This protocol, which can be completed in 7 h, allows the assessment of quantitative changes in SEZ NSCs and the examination of their molecular and functional characteristics.
AU  - Fischer, J.
AU  - Beckervordersandforth, R.
AU  - Tripathi, P.
AU  - Steiner-Mezzadri, A.
AU  - Ninkovic, J.
AU  - Götz, M.
C1  - 6792
C2  - 29278
SP  - 1981-1989
TI  - Prospective isolation of adult neural stem cells from the mouse subependymal zone.
JO  - Nat. Protoc.
VL  - 6
IS  - 12
PB  - nature publishing group
PY  - 2011
SN  - 1754-2189
ER  - 

TY  - JOUR
AB  - Instructing glial cells to generate neurons may prove a strategy to replace neurons that have degenerated. Here we describe a robust protocol for the efficient in vitro conversion of postnatal astroglia from the murine cerebral cortex into functional, synapse-forming neurons. This protocol involves two steps: (i) expansion of astroglial cells (7 days) and (ii) astroglia-to-neuron conversion induced by persistent and strong retroviral expression of Neurogenin2 or Mash1 and/or Dlx2 for generation of glutamatergic or GABAergic neurons, respectively (7-21 days for different degrees of maturity). Our protocol of astroglia-to-neuron conversion by a single neurogenic transcription factor provides a stringent experimental system to study the specification of a selective neuronal subtype, thus offering an alternative to the use of embryonic or neural stem cells. Moreover it can be a useful model for studies of lineage conversion from non-neuronal cells with potential for brain regenerative medicine.
AU  - Heinrich, C.*
AU  - Gascón, S.
AU  - Masserdotti, G.*
AU  - Lepier, A.*
AU  - Sánchez, R.*
AU  - Simon-Ebert, T.*
AU  - Schroeder, T.
AU  - Götz, M.
AU  - Berninger, B.
C1  - 5935
C2  - 28369
SP  - 214-228
TI  - Generation of subtype-specific neurons from postnatal astroglia of the mouse cerebral cortex.
JO  - Nat. Protoc.
VL  - 6
IS  - 2
PB  - Nature Publ. Group
PY  - 2011
SN  - 1754-2189
ER  - 

TY  - JOUR
AB  - A comprehensive understanding of the cell biology of adult neural stem cells (aNSCs) requires direct observation of aNSC division and lineage progression in the absence of niche-dependent signals. Here we describe a culture preparation of the adult mouse subependymal zone (SEZ), which allows for continuous single-cell tracking of aNSC behavior. The protocol involves the isolation (∼3 h) and culture of cells from the adult SEZ at low density in the absence of mitogenic growth factors in chemically defined medium and subsequent live imaging using time-lapse video microscopy (5-7 d); these steps are followed by postimaging immunocytochemistry to identify progeny (∼7 h). This protocol enables the observation of the progression from slow-dividing aNSCs of radial/astroglial identity up to the neuroblast stage, involving asymmetric and symmetric cell divisions of distinct fast-dividing precursors. This culture provides an experimental system for studying instructive or permissive effects of signal molecules on aNSC modes of cell division and lineage progression.
AU  - Ortega, F.*
AU  - Costa, M.R.*
AU  - Simon-Ebert, T.*
AU  - Schroeder, T.
AU  - Götz, M.
AU  - Berninger, B.
C1  - 6692
C2  - 29129
SP  - 1847-1859
TI  - Using an adherent cell culture of the mouse subependymal zone to study the behavior of adult neural stem cells on a single-cell level.
JO  - Nat. Protoc.
VL  - 6
IS  - 12
PB  - Nature America
PY  - 2011
SN  - 1754-2189
ER  - 

TY  - JOUR
AB  - Multispectral optoacoustic tomography (MSOT) has recently been developed to enable visualization of optical contrast and tissue biomarkers, with resolution and speed representative of ultrasound. In the implementation described here, MSOT enables operation in real-time mode by capturing single cross-sectional images in <1 ms from living small animals (e.g., mice) and other tissues of similar dimensions. At the core of the method is illumination of the object using multiple wavelengths in order to resolve spectrally distinct biomarkers over background tissue chromophores. The system allows horizontal placement of a mouse in the imaging chamber and three-dimensional scanning of the entire body without the need to immerse the mouse in water. Here we provide a detailed description of the MSOT scanner components, system calibration, selection of image reconstruction algorithms and animal handling. Overall, the entire protocol can be completed within 15-30 min for acquisition of a whole-body multispectral data set from a living mouse.
AU  - Razansky, D.
AU  - Bühler, A.
AU  - Ntziachristos, V.
C1  - 6552
C2  - 28872
SP  - 1121-1129
TI  - Volumetric real-time multispectral optoacoustic tomography of biomarkers.
JO  - Nat. Protoc.
VL  - 6
IS  - 8
PB  - Nature Publ. Group
PY  - 2011
SN  - 1754-2189
ER  - 

TY  - JOUR
AB  - Steroids are powerful signalling molecules regulating a variety of physiological processes such as cell proliferation, cell differentiation, and reproduction. Variations in steroid concentrations may hint at hormonal imbalances or metabolic disorders that allow for a diagnosis of human diseases. The measurement of steroid hormone concentrations is relevant to basic research as well as clinical sciences. For example, the systematic generation of mouse mutants has led to a need for comprehensive phenotyping. As these mouse models may bear defects that affect steroid synthesis or homeostasis, knowledge of the mouse steroid metabolome could help to identify these mutants and connect phenotypes to their molecular cause 1.Metabolic characterisation of a large human cohort recently led to the discovery of associations between phenotype, metabolome, and genotype as shown by us 2. To expand our knowledge of the metabolome to other substance classes, a high-throughput, robust, sensitive and specific steroid phenotyping method is required. For several years, immunoassays have been the standard method for measuring steroid concentrations. Recently, liquid chromatography tandem mass spectrometry methods for steroid analysis have been developed that combine the advantages of immunoassays (i.e. sensitivity) with the specificity of mass spectrometry 3. This protocol describes a high-throughput method to analyze several different steroids in plasma. By using online-SPE coupled to LC-MS/MS, six different steroids (androstenedione, testosterone, cortisone, cortisol, progesterone, 17OH-progesterone) are readily quantifiable in human plasma, while three steroids (androstenedione, testosterone, corticosterone) can be quantified in mouse plasma. The method requires a minimum of hands-on time by the experimenter while simultaneously providing concentrations of several interesting steroids.
AU  - Haller, F.
AU  - Prehn, C.
AU  - Adamski, J.
C1  - 17153
C2  - 30851
SP  - DOI: 10.1038/nprot.2010.22
TI  - Quantification of steroids in human and mouse plasma using online solid phase extraction coupled to liquid chromatography tandem mass spectrometry.
JO  - Nat. Protoc.
PB  - Nature Publishing
PY  - 2010
SN  - 1754-2189
ER  - 

TY  - JOUR
AB  - Stable-isotope probing is a method used in microbial ecology that provides a means by which specific functional groups of organisms that incorporate particular substrates are identified without the prerequisite of cultivation. Stable-isotope-labeled carbon (13C) or nitrogen (15N) sources are assimilated into microbial biomass of environmental samples. Separation and molecular analysis of labeled nucleic acids (DNA or RNA) reveals phylogenetic and functional information about the microorganisms responsible for the metabolism of a particular substrate. Here, we highlight general guidelines for incubating environmental samples with labeled substrate and provide a detailed protocol for separating labeled DNA from unlabeled community DNA. The protocol includes a modification of existing published methods, which maximizes the recovery of labeled DNA from CsCl gradients. The separation of DNA and retrieval of unlabeled and labeled fractions can be performed in 4–5 days, with much of the time being committed to the ultracentrifugation step.
AU  - Neufeld, J.D.*
AU  - Vohra, J.*
AU  - Dumont, M.G.*
AU  - Lüders, T.
AU  - Manefield, M.*
AU  - Friedrich, M.W.*
AU  - Murrell, J.C.*
C1  - 5815
C2  - 24425
SP  - 860-866
TI  - DNA stable-isotope probing.
JO  - Nat. Protoc.
VL  - 2
IS  - 4
PB  - Nature Publ. Group
PY  - 2007
SN  - 1754-2189
ER  - 

TY  - JOUR
AB  - At the heart of microbial ecology lies a true scientific dichotomy. On the one hand, we know microbes are responsible for processes on which all other life on Earth is dependent; their removal would mean the cessation of all known life. However, in opposition, the majority of extant microbial species in natural environments have never been cultured or studied in a laboratory as living organisms. Owing to these factors, the question of "who does what?" has been a major barrier to understanding how microbially mediated ecosystem level events occur. Recently, the use of stable isotopes (13C) to trace carbon from specific substrates into microbes that assimilate carbon from that substrate has significantly advanced our understanding of the relationship between environmental processes and microbial phylogeny.
AU  - Whiteley, A.S.*
AU  - Thomson, B.*
AU  - Lüders, T.
AU  - Manefield, M.*
C1  - 1759
C2  - 24426
SP  - 838-844
TI  - RNA stable-isotope probing.
JO  - Nat. Protoc.
VL  - 2
IS  - 4
PB  - Nature Publ. Group
PY  - 2007
SN  - 1754-2189
ER  -