Browse Articles

  • Protocol |

    Ice formation has hindered organ preservation below 0 °C. In this protocol, human livers are preconditioned with cryoprotective agents during machine perfusion and then supercooled to avoid ice formation.

    • Reinier J. de Vries
    • , Shannon N. Tessier
    • , Peony D. Banik
    • , Sonal Nagpal
    • , Stephanie E. J. Cronin
    • , Sinan Ozer
    • , Ehab O. A. Hafiz
    • , Thomas M. van Gulik
    • , Martin L. Yarmush
    • , James F. Markmann
    • , Mehmet Toner
    • , Heidi Yeh
    •  & Korkut Uygun
  • Protocol |

    This protocol details the assembly and use of the wide field-of-view nematode tracking platform (WF-NTP), which enables the simultaneous analysis of thousands of worms with respect to multiple behavioral parameters.

    • Mandy Koopman
    • , Quentin Peter
    • , Renée I. Seinstra
    • , Michele Perni
    • , Michele Vendruscolo
    • , Christopher M. Dobson
    • , Tuomas P. J. Knowles
    •  & Ellen A. A. Nollen
  • Protocol |

    This protocol describes how to construct and use an open-source robot for automatically performing cranial microsurgeries such as skull thinning and various-sized craniotomies in mice.

    • Mathew L. Rynes
    • , Leila Ghanbari
    • , Daniel Sousa Schulman
    • , Samantha Linn
    • , Michael Laroque
    • , Judith Dominguez
    • , Zahra S. Navabi
    • , Peter Sherman
    •  & Suhasa B. Kodandaramaiah
  • Protocol |

    High-resolution structural analysis of macromolecular complexes by cryo-ET requires extremely thin samples. This protocol describes how to prepare thin specimens using FIB milling from frozen cells on grids, which enables direct structural analysis of biomolecules in their native environments, i.e., cells.

    • Felix R. Wagner
    • , Reika Watanabe
    • , Ruud Schampers
    • , Digvijay Singh
    • , Hans Persoon
    • , Miroslava Schaffer
    • , Peter Fruhstorfer
    • , Jürgen Plitzko
    •  & Elizabeth Villa
  • Protocol |

    Global Natural Product Social Molecular Networking (GNPS) is an online tandem mass spectrometry (MS2) data curation and analysis infrastructure. This protocol describes how to use GNPS to explore uploaded metabolomics data.

    • Allegra T. Aron
    • , Emily C. Gentry
    • , Kerry L. McPhail
    • , Louis-Félix Nothias
    • , Mélissa Nothias-Esposito
    • , Amina Bouslimani
    • , Daniel Petras
    • , Julia M. Gauglitz
    • , Nicole Sikora
    • , Fernando Vargas
    • , Justin J. J. van der Hooft
    • , Madeleine Ernst
    • , Kyo Bin Kang
    • , Christine M. Aceves
    • , Andrés Mauricio Caraballo-Rodríguez
    • , Irina Koester
    • , Kelly C. Weldon
    • , Samuel Bertrand
    • , Catherine Roullier
    • , Kunyang Sun
    • , Richard M. Tehan
    • , Cristopher A. Boya P.
    • , Martin H. Christian
    • , Marcelino Gutiérrez
    • , Aldo Moreno Ulloa
    • , Javier Andres Tejeda Mora
    • , Randy Mojica-Flores
    • , Johant Lakey-Beitia
    • , Victor Vásquez-Chaves
    • , Yilue Zhang
    • , Angela I. Calderón
    • , Nicole Tayler
    • , Robert A. Keyzers
    • , Fidele Tugizimana
    • , Nombuso Ndlovu
    • , Alexander A. Aksenov
    • , Alan K. Jarmusch
    • , Robin Schmid
    • , Andrew W. Truman
    • , Nuno Bandeira
    • , Mingxun Wang
    •  & Pieter C. Dorrestein
  • Protocol |

    The authors demonstrate two variants of the cellular thermal shift assay (CETSA) protocol—the melt curve and the isothermal dose–response—for use on intact cell or lysate P. falciparum samples for identification of antimalarial drug targets.

    • Jerzy Michal Dziekan
    • , Grennady Wirjanata
    • , Lingyun Dai
    • , Ka Diam Go
    • , Han Yu
    • , Yan Ting Lim
    • , Liyan Chen
    • , Loo Chien Wang
    • , Brenda Puspita
    • , Nayana Prabhu
    • , Radoslaw M. Sobota
    • , Pär Nordlund
    •  & Zbynek Bozdech
  • Protocol |

    The authors describe DISCOVER-seq, a method to detect off-targets of CRISPR–Cas genome editing based on ChIP-seq analysis of MRE11 recruitment to DSBs, and subsequent bioinformatics analysis of sequencing data using the BLENDER pipeline.

    • Beeke Wienert
    • , Stacia K. Wyman
    • , Charles D. Yeh
    • , Bruce R. Conklin
    •  & Jacob E. Corn
  • Protocol |

    neuECG is the simultaneous noninvasive recording of ECG and skin sympathetic nerve activity and thus can directly record sympathetic nerve activity over a long period of time without the need for invasive procedures.

    • Takashi Kusayama
    • , Johnson Wong
    • , Xiao Liu
    • , Wenbo He
    • , Anisiia Doytchinova
    • , Eric A. Robinson
    • , David E. Adams
    • , Lan S. Chen
    • , Shien-Fong Lin
    • , Katherine Davoren
    • , Ronald G. Victor
    • , Cheng Cai
    • , Ming-Yan Dai
    • , Ying Tian
    • , Pei Zhang
    • , Dereen Ernst
    • , Richard H. Rho
    • , Minglong Chen
    • , Yong-Mei Cha
    • , David R. Walega
    • , Thomas H. Everett IV
    •  & Peng-Sheng Chen
  • Protocol |

    Here, the authors describe an international consensus procedure for determining freely dissolved concentrations (Cfree) of hydrophobic organic chemicals in sediment and soil samples by passive sampling.

    • Michiel T. O. Jonker
    • , Robert M. Burgess
    • , Upal Ghosh
    • , Philip M. Gschwend
    • , Sarah E. Hale
    • , Rainer Lohmann
    • , Michael J. Lydy
    • , Keith A. Maruya
    • , Danny Reible
    •  & Foppe Smedes
  • Protocol |

    Electrocatalysis is now being used for many oxidation reactions in organic synthesis. This protocol describes how to set up an electrolytic cell for use in cobalt-catalyzed C–H/N–H alkyne annulations and C–H oxygenations.

    • Cong Tian
    • , Tjark H. Meyer
    • , Maximilian Stangier
    • , Uttam Dhawa
    • , Karsten Rauch
    • , Lars H. Finger
    •  & Lutz Ackermann
  • Protocol |

    The HDOCK server is developed for template-based and template-free protein–protein docking, using amino acid sequences or PDB structures as inputs. HDOCK can incorporate SAXS data and can be applied to protein–RNA/DNA docking.

    • Yumeng Yan
    • , Huanyu Tao
    • , Jiahua He
    •  & Sheng-You Huang
  • Protocol |

    [18F]6-fluoro-l-DOPA ([18F]FDOPA) is used for diagnostic PET imaging. Using this protocol, radiofluorination of the electron-rich catechol ring can be achieved in the presence of the amino acid group via Cu-mediated fluorination of a pinacol boronate precursor.

    • Andrew V. Mossine
    • , Sean S. Tanzey
    • , Allen F. Brooks
    • , Katarina J. Makaravage
    • , Naoko Ichiishi
    • , Jason M. Miller
    • , Bradford D. Henderson
    • , Thomas Erhard
    • , Christian Bruetting
    • , Marc B. Skaddan
    • , Melanie S. Sanford
    •  & Peter J. H. Scott
  • Protocol |

    This protocol describes in vitro procedures for generation of synthetic single-domain antibodies called ‘sybodies’. Sybodies can be engineered to target specific protein conformations, labile membrane proteins or protein complexes.

    • Iwan Zimmermann
    • , Pascal Egloff
    • , Cedric A. J. Hutter
    • , Benedikt T. Kuhn
    • , Philipp Bräuer
    • , Simon Newstead
    • , Roger J. P. Dawson
    • , Eric R. Geertsma
    •  & Markus A. Seeger
  • Protocol |

    Size-selected and amplified circular DNA molecules are sequenced on the PacBio platform and processed with a custom pipeline, resulting in full-length annotated genomes of circular DNA viruses and sequences of extrachromosomal circular DNA at single-molecule resolution.

    • Devang Mehta
    • , Luc Cornet
    • , Matthias Hirsch-Hoffmann
    • , Syed Shan-e-Ali Zaidi
    •  & Hervé Vanderschuren
  • Protocol |

    Libraries of highly immature, ground-state human intestinal stem cells are established from endoscopic biopsy samples. Single cell–derived clones can be rapidly expanded for molecular genetics, 3D differentiation, disease modeling and drug discovery.

    • Marcin Duleba
    • , Yusuke Yamamoto
    • , Rahul Neupane
    • , Wei Rao
    • , Jingzhong Xie
    • , Yutao Qi
    • , Audrey-Ann Liew
    • , Suchan Niroula
    • , Yanting Zhang
    • , Rajasekaran Mahalingam
    • , Shan Wang
    • , Kristina Goller
    • , Jaffer A. Ajani
    • , Matthew Vincent
    • , Khek Yu Ho
    • , Jason K. Hou
    • , Jeffrey S. Hyams
    • , Francisco A. Sylvester
    • , Christopher P. Crum
    • , Frank McKeon
    •  & Wa Xian
  • Protocol |

    Paraffin-embedded, fresh-frozen or chemically stained fixed human tissues are isotropically expanded by 4–5× in linear dimension to enable nanoscale-resolution imaging on conventional microscopes.

    • Octavian Bucur
    • , Feifei Fu
    • , Mike Calderon
    • , Geetha H. Mylvaganam
    • , Ngoc L. Ly
    • , Jimmy Day
    • , Simon Watkin
    • , Bruce D. Walker
    • , Edward S. Boyden
    •  & Yongxin Zhao
  • Protocol |

    Native mass spectrometry is a powerful technique for studying intact proteins and protein complexes. Using this protocol, meaningful structural information on protein complexes ejected from sonicated native membrane vesicles can be obtained.

    • Dror S. Chorev
    • , Haiping Tang
    • , Sarah L. Rouse
    • , Jani Reddy Bolla
    • , Andriko von Kügelgen
    • , Lindsay A. Baker
    • , Di Wu
    • , Joseph Gault
    • , Kay Grünewald
    • , Tanmay A. M. Bharat
    • , Stephen J. Matthews
    •  & Carol V. Robinson
  • Protocol |

    In this protocol, mice are inoculated with two separate tumors derived from the same cell line. One tumor is removed and assessed before treatment; the other is used to assess the effect of treatment.

    • Rachael M. Zemek
    • , Vanessa S. Fear
    • , Cath Forbes
    • , Emma de Jong
    • , Thomas H. Casey
    • , Louis Boon
    • , Timo Lassmann
    • , Anthony Bosco
    • , Michael J. Millward
    • , Anna K. Nowak
    • , Richard A. Lake
    •  & W. Joost Lesterhuis
  • Review Article |

    This tutorial and the accompanying poster (https://doi.org/10.1038/s41596-020-0307-7) provide a guide for performing quantitative fluorescence imaging using confocal microscopy. It includes advice and troubleshooting information from sample preparation and microscope setup to data analysis and statistics.

    • James Jonkman
    • , Claire M. Brown
    • , Graham D. Wright
    • , Kurt I. Anderson
    •  & Alison J. North
  • Protocol |

    This protocol describes how to differentiate human induced pluripotent stem cells into oogonia in vitro. It is suitable for investigating the mechanisms of human primordial germ cell specification and epigenetic reprogramming.

    • Chika Yamashiro
    • , Kotaro Sasaki
    • , Shihori Yokobayashi
    • , Yoji Kojima
    •  & Mitinori Saitou
  • Protocol |

    In this operant model, rats press a lever to obtain addictive drugs or rewarding social interaction with a peer. The model can thus be used to study the role of operant social reward in addiction and addiction vulnerability in the context of choices.

    • Marco Venniro
    •  & Yavin Shaham
  • Protocol |

    Reductive amination is essential to the preparation of amines (e.g., pharmaceuticals and industrial products). This protocol shows how to prepare and use graphitic shell–encapsulated cobalt-based nanoparticles as catalysts for this reaction.

    • Kathiravan Murugesan
    • , Vishwas G. Chandrashekhar
    • , Thirusangumurugan Senthamarai
    • , Rajenahally V. Jagadeesh
    •  & Matthias Beller
  • Protocol |

    This protocol describes the generation and delivery of embedded viral barcode libraries to track single-cell clones. Barcodes are amplified from genomic DNA and quantified by high-throughput sequencing and bioinformatics analysis.

    • Charles Bramlett
    • , Du Jiang
    • , Anna Nogalska
    • , Jiya Eerdeng
    • , Jorge Contreras
    •  & Rong Lu
  • Protocol |

    Automation of new methods for 18F incorporation into unactivated (hetero)arenes for translation to the clinic has progressed slowly. This protocol describes a workflow leading to automated radiosynthesis of the PARP inhibitor [18F]olaparib.

    • Florian Guibbal
    • , Patrick G. Isenegger
    • , Thomas C. Wilson
    • , Anna Pacelli
    • , Damien Mahaut
    • , Jeroen B. I. Sap
    • , Nicholas J. Taylor
    • , Stefan Verhoog
    • , Sean Preshlock
    • , Rebekka Hueting
    • , Bart Cornelissen
    •  & Véronique Gouverneur
  • Protocol |

    R-DeeP identifies, quantifies and analyzes RNA-dependent proteins using sucrose density gradient ultracentrifugation followed by western blotting for individual proteins or proteome-wide mass spectrometry and biostatistical analysis. RNase-treated samples are compared with controls to reveal differences in protein complex composition in the presence of RNA.

    • Maiwen Caudron-Herger
    • , Elsa Wassmer
    • , Isha Nasa
    • , Astrid-Solveig Schultz
    • , Jeanette Seiler
    • , Arminja N. Kettenbach
    •  & Sven Diederichs
  • Protocol |

    This protocol describes collection, storage and lysis of different types of mouse organs for monitoring gene expression on a whole-organism scale. Procedures for high-throughput RNA extraction and multiplexed RNA sequencing library preparation are included.

    • Surya Pandey
    • , Michihiro Takahama
    • , Adam Gruenbaum
    • , Makda Zewde
    • , Katerina Cheronis
    •  & Nicolas Chevrier
  • Protocol |

    This protocol describes how to visualize and quantify translation of individual mRNAs in live cells by fluorescence microscopy. Example procedures for quantitative analysis of 3ʹ-UTR translation, nonsense-mediated mRNA decay, and translation start-site selection are provided.

    • Deepak Khuperkar
    • , Tim A. Hoek
    • , Stijn Sonneveld
    • , Bram M. P. Verhagen
    • , Sanne Boersma
    •  & Marvin E. Tanenbaum
  • Protocol |

    This protocol presents an imaging system that uses high-resolution confocal microscopy with a fluorochrome-labeled avidin probe to visualize mast cell degranulation in single cells subjected to shRNA knockdown or CRISPR–Cas9 gene editing.

    • Jelle Folkerts
    • , Nicolas Gaudenzio
    • , Marcus Maurer
    • , Rudi W. Hendriks
    • , Ralph Stadhouders
    • , See-Ying Tam
    •  & Stephen J. Galli
  • Protocol |

    This protocol describes how to establish an ovine critical-size, segmental bone defect model to study bone regeneration and reconstruction.

    • David S. Sparks
    • , Siamak Saifzadeh
    • , Flavia Medeiros Savi
    • , Constantin E. Dlaska
    • , Arne Berner
    • , Jan Henkel
    • , Johannes C. Reichert
    • , Martin Wullschleger
    • , Jiongyu Ren
    • , Amaia Cipitria
    • , Jacqui A. McGovern
    • , Roland Steck
    • , Michael Wagels
    • , Maria Ann Woodruff
    • , Michael A. Schuetz
    •  & Dietmar W. Hutmacher
  • Protocol |

    This protocol describes the instrumentation and analysis methods needed to quantify particle dynamics during new particle formation of sub-3-nm aerosol particles in chamber experiments.

    • Lubna Dada
    • , Katrianne Lehtipalo
    • , Jenni Kontkanen
    • , Tuomo Nieminen
    • , Rima Baalbaki
    • , Lauri Ahonen
    • , Jonathan Duplissy
    • , Chao Yan
    • , Biwu Chu
    • , Tuukka Petäjä
    • , Kari Lehtinen
    • , Veli-Matti Kerminen
    • , Markku Kulmala
    •  & Juha Kangasluoma
  • Protocol |

    This protocol describes how to anesthetize and image different organs of Drosophila larvae, including epidermis, gut, imaginal discs, neurons, fat body, tracheae, muscles and hemocytes. It also explains how to use laser ablation to probe cell properties.

    • Parisa Kakanj
    • , Sabine A. Eming
    • , Linda Partridge
    •  & Maria Leptin
  • Protocol Extension |

    This Protocol Extension describes how to perform formaldehyde-assisted isolation of regulatory elements (FAIRE) from Arabidopsis leaves. The FAIRE protocol is optimized for compatibility with downstream qPCR analysis and next-generation sequencing.

    • Stephani Baum
    • , Eva-Maria Reimer-Michalski
    • , Michal R. Jaskiewicz
    •  & Uwe Conrath
  • Protocol |

    Mice are trained to use a custom-made robotic manipulandum to enable study of motor learning. The device can be used in conjunction with neurophysiological and microscopy techniques that require head fixation.

    • Mark J. Wagner
    • , Joan Savall
    • , Tony Hyun Kim
    • , Mark J. Schnitzer
    •  & Liqun Luo
  • Protocol Extension |

    In this extension to their original protocol applying TAR cloning to mammalian genomes, the authors apply the technique to microbes and environmental DNA samples, by adding ARS-like elements not commonly found in microbial genomes to the TAR cloning vector.

    • Natalay Kouprina
    • , Vladimir N. Noskov
    •  & Vladimir Larionov
  • Protocol |

    Various types of endothelial cells and CD157+ vascular-resident endothelial stem cells are isolated from mice by mechano-enzymatic tissue digestion followed by fluorescence-activated cell sorting.

    • Hisamichi Naito
    • , Taku Wakabayashi
    • , Masako Ishida
    • , Chang-Hyun Gil
    • , Tomohiro Iba
    • , Fitriana Nur Rahmawati
    • , Shota Shimizu
    • , Mervin C. Yoder
    •  & Nobuyuki Takakura
  • Protocol |

    This Protocol describes how to perform online buffer exchange (OBE) coupled to native mass spectrometry (nMS) for rapid screening of structural features of pre-purified proteins, protein complexes or clarified cell lysates.

    • Zachary L. VanAernum
    • , Florian Busch
    • , Benjamin J. Jones
    • , Mengxuan Jia
    • , Zibo Chen
    • , Scott E. Boyken
    • , Aniruddha Sahasrabuddhe
    • , David Baker
    •  & Vicki H. Wysocki