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Nature Protocols

Yuri M Efremov, Alexander X Cartagena-Rivera, Ahmad I M Athamneh, Daniel M Suter, Arvind Raman
The goal of mechanobiology is to understand the links between changes in the physical properties of living cells and normal physiology and disease. This requires mechanical measurements that have appropriate spatial and temporal resolution within a single cell. Conventional atomic force microscopy (AFM) methods that acquire force curves pointwise are used to map the heterogeneous mechanical properties of cells. However, the resulting map acquisition time is much longer than that required to study many dynamic cellular processes...
September 14, 2018: Nature Protocols
Evelina Tutucci, Maria Vera, Robert H Singer
The MS2 system has been widely used, in organisms ranging from bacteria to higher eukaryotes, to image single mRNAs in intact cells with high precision. We have recently re-engineered the MS2 system for accurate detection of mRNAs in living Saccharomyces cerevisiae. Previous MS2 systems affected the degradation of the tagged mRNA, which led to accumulation of MS2 fragments and to erroneous conclusions about mRNA localization and expression. Here we describe a step-by-step protocol for the use of our latest MS2 system (MBSV6) for detecting endogenously tagged mRNAs using wide-field fluorescent microscopy in living yeast...
September 14, 2018: Nature Protocols
Julie Marchesan, Mustafa S Girnary, Li Jing, Michael Zhe Miao, Shaoping Zhang, Lu Sun, Thiago Morelli, Mark H Schoenfisch, Naohiro Inohara, Steven Offenbacher, Yizu Jiao
Periodontal disease (PD) is a common dental disease associated with the interaction between dysbiotic oral microbiota and host immunity. It is a prevalent disease, resulting in loss of gingival tissue, periodontal ligament, cementum and alveolar bone. PD is a major form of tooth loss in the adult population. Experimental animal models have enabled the study of PD pathogenesis and are used to test new therapeutic approaches for treating the disease. The ligature-induced periodontitis model has several advantages as compared with other models, including rapid disease induction, predictable bone loss and the capacity to study periodontal tissue and alveolar bone regeneration because the model is established within the periodontal apparatus...
September 14, 2018: Nature Protocols
Steven A Sloan, Jimena Andersen, Anca M Pașca, Fikri Birey, Sergiu P Pașca
The ability to generate region-specific three-dimensional (3D) models to study human brain development offers great promise for understanding the nervous system in both healthy individuals and patients. In this protocol, we describe how to generate and assemble subdomain-specific forebrain spheroids, also known as brain region-specific organoids, from human pluripotent stem cells (hPSCs). We describe how to pattern the neural spheroids toward either a dorsal forebrain or a ventral forebrain fate, establishing human cortical spheroids (hCSs) and human subpallial spheroids (hSSs), respectively...
September 10, 2018: Nature Protocols
Florian A Dehmelt, Adam von Daranyi, Claire Leyden, Aristides B Arrenberg
The version of this paper originally published contained the following text errors: (1) In the abstract, "(ii) visual stimulation with moving bars; (ii) eye detection and tracking, as well as general motion detection" should have been "(ii) visual stimulation with moving bars; (iii) eye detection and tracking, as well as general motion detection." (2) In the legend for Table 1, "vertical pixel coordinate; LE, left eye; RE, right eye; x, horizontal pixel coordinate; y" should have read "LE, left eye; RE, right eye; x, horizontal pixel coordinate; y, vertical pixel coordinate...
August 21, 2018: Nature Protocols
Tommi Huhtamäki, Xuelin Tian, Juuso T Korhonen, Robin H A Ras
The version of this Protocol originally published contained typographical errors that affected the accuracy/readability of the text. In Fig. 4e, the line "Contact angle remainsstable" should have read "Contact angle remains stable." In Table 1, in the "Advantages" column, the second instance of "Simple" was incorrectly associated with the "Sessile-drop goniometry" method; it should have corresponded to the "Tilting plate" method. In Table 2, in the "Issues" column, the entry "Difficult to place baseline when the RCA is ~90°" was broken incorrectly in a way that might have suggested that "the RCA is ~90°" was a separate issue...
August 7, 2018: Nature Protocols
Ricardo Cruz-Acuña, Miguel Quirós, Sha Huang, Dorothée Siuda, Jason R Spence, Asma Nusrat, Andrés J García
In vitro differentiation of human pluripotent stem cell (hPSC)-derived organoids (HOs) facilitates the production of multicellular three-dimensional structures analogous to native human tissues. Most current methods for the generation of HOs rely on Matrigel, a poorly defined basement membrane derivative secreted by Engelbreth-Holm-Swarm mouse sarcoma cells, limiting the potential use of HOs for regenerative medicine applications. Here, we describe a protocol for the synthesis of a fully defined, synthetic hydrogel that supports the generation and culture of HOs...
September 2018: Nature Protocols
Marco Capogrosso, Fabien B Wagner, Jerome Gandar, Eduardo Martin Moraud, Nikolaus Wenger, Tomislav Milekovic, Polina Shkorbatova, Natalia Pavlova, Pavel Musienko, Erwan Bezard, Jocelyne Bloch, Grégoire Courtine
Epidural electrical stimulation (EES) of the spinal cord and real-time processing of gait kinematics are powerful methods for the study of locomotion and the improvement of motor control after injury or in neurological disorders. Here, we describe equipment and surgical procedures that can be used to acquire chronic electromyographic (EMG) recordings from leg muscles and to implant targeted spinal cord stimulation systems that remain stable up to several months after implantation in rats and nonhuman primates...
September 2018: Nature Protocols
Sean J Humphrey, Ozge Karayel, David E James, Matthias Mann
Mass spectrometry has transformed the field of cell signaling by enabling global studies of dynamic protein phosphorylation ('phosphoproteomics'). Recent developments are enabling increasingly sophisticated phosphoproteomics studies, but practical challenges remain. The EasyPhos workflow addresses these and is sufficiently streamlined to enable the analysis of hundreds of phosphoproteomes at a depth of >10,000 quantified phosphorylation sites. Here we present a detailed and updated workflow that further ensures high performance in sample-limited conditions while also reducing sample preparation time...
September 2018: Nature Protocols
Darren Rodenhizer, Teresa Dean, Bin Xu, Dan Cojocari, Alison P McGuigan
This protocol describes how to build and implement a three-dimensional (3D) cell culture system, TRACER (tissue roll for analysis of cellular environment and response), that enables analysis of cellular behavior and phenotype in hypoxic gradients. TRACER consists of infiltrating cells encapsulated in a hydrogel extracellular matrix (ECM) within a thin strip of porous cellulose scaffolding that is then rolled around an oxygen-impermeable mandrel for assembly of thick and layered 3D tissue constructs that develop cell-defined oxygen gradients...
September 2018: Nature Protocols
Divya Mohan, Daniel L Wansley, Brandon M Sie, Muhammad S Noon, Alan N Baer, Uri Laserson, H Benjamin Larman
The binding specificities of an individual's antibody repertoire contain a wealth of biological information. They harbor evidence of environmental exposures, allergies, ongoing or emerging autoimmune disease processes, and responses to immunomodulatory therapies, for example. Highly multiplexed methods to comprehensively interrogate antibody-binding specificities have therefore emerged in recent years as important molecular tools. Here, we provide a detailed protocol for performing 'phage immunoprecipitation sequencing' (PhIP-Seq), which is a powerful method for analyzing antibody-repertoire binding specificities with high throughput and at low cost...
September 2018: Nature Protocols
Milka Doktorova, Frederick A Heberle, Barbara Eicher, Robert F Standaert, John Katsaras, Erwin London, Georg Pabst, Drew Marquardt
Freely suspended liposomes are widely used as model membranes for studying lipid-lipid and protein-lipid interactions. Liposomes prepared by conventional methods have chemically identical bilayer leaflets. By contrast, living cells actively maintain different lipid compositions in the two leaflets of the plasma membrane, resulting in asymmetric membrane properties that are critical for normal cell function. Here, we present a protocol for the preparation of unilamellar asymmetric phospholipid vesicles that better mimic biological membranes...
September 2018: Nature Protocols
Suman Ranjit, Leonel Malacrida, David M Jameson, Enrico Gratton
Fluorescence lifetime imaging microscopy (FLIM) is used in diverse disciplines, including biology, chemistry and biophysics, but its use has been limited by the complexity of the data analysis. The phasor approach to FLIM has the potential to markedly reduce this complexity and at the same time provide a powerful visualization of the data content. Phasor plots for fluorescence lifetime analysis were originally developed as a graphical representation of excited-state fluorescence lifetimes for in vitro systems...
September 2018: Nature Protocols
Xin Zhang, Yang He, Jia Liu, Mark E Bowden, Libor Kovarik, Scott X Mao, Chongmin Wang, James J De Yoreo, Kevin M Rosso
Biominerals serve as critical structures of living systems and play important roles in biochemical processes. Understanding their crystallization mechanisms is therefore central to many areas of biology, biogeoscience, and biochemistry. Some biominerals, such as bone and dentin, are hierarchical nanocomposite structures constructed by sequential addition of individual oriented nanocrystals. The driving forces that enable this oriented assembly are still poorly understood, with advances in understanding limited in part by the availability of techniques that can precisely measure the delicate interactions between nanocrystals as a function of their separation distance and mutual orientation...
September 2018: Nature Protocols
Boyang Zhang, Benjamin Fook Lun Lai, Ruoxiao Xie, Locke Davenport Huyer, Miles Montgomery, Milica Radisic
Microengineered biomimetic systems for organ-on-a-chip or tissue engineering purposes often fail as a result of an inability to recapitulate the in vivo environment, specifically the presence of a well-defined vascular system. To address this limitation, we developed an alternative method to cultivate three-dimensional (3D) tissues by incorporating a microfabricated scaffold, termed AngioChip, with a built-in perfusable vascular network. Here, we provide a detailed protocol for fabricating the AngioChip scaffold, populating it with endothelial cells and parenchymal tissues, and applying it in organ-on-a-chip drug testing in vitro and surgical vascular anastomosis in vivo...
August 2018: Nature Protocols
Yongfu Wang, Zulin Yu, Cori K Cahoon, Tari Parmely, Nancy Thomas, Jay R Unruh, Brian D Slaughter, R Scott Hawley
Biologists have long been fascinated with the organization and function of intricate protein complexes. Therefore, techniques for precisely imaging protein complexes and the location of proteins within these complexes are critically important and often require multidisciplinary collaboration. A challenge in these explorations is the limited resolution of conventional light microscopy. However, a new microscopic technique has circumvented this resolution limit by making the biological sample larger, thus allowing for super-resolution of the enlarged structure...
August 2018: Nature Protocols
Annabelle C Singer, Anthony J Martorell, J Miller Douglas, Fatema Abdurrob, Matthew K Attokaren, John Tipton, Hansruedi Mathys, Chinnakkaruppan Adaikkan, Li-Huei Tsai
Microglia, the primary immune cells of the brain, play a key role in pathological and normal brain function. Growing efforts aim to reveal how these cells may be harnessed to treat both neurodegenerative diseases such as Alzheimer's and developmental disorders such as schizophrenia and autism. We recently showed that using noninvasive exposure to 40-Hz white-light (4,000 K) flicker to drive 40-Hz neural activity transforms microglia into an engulfing state and reduces amyloid beta, a peptide thought to initiate neurotoxic events in Alzheimer's disease (AD)...
August 2018: Nature Protocols
Niankai Fu, Gregory S Sauer, Song Lin
This protocol describes an electrochemical synthesis of 1,2-diazides from alkenes. Organic azides are highly versatile intermediates for synthetic chemistry, materials, and biological applications. 1,2-Diazides are commonly reduced to form 1,2-diamines, which are prevalent structural motifs in bioactive natural products, therapeutic agents, and molecular catalysts. The electrochemical formation of 1,2-diazides involves the anodic generation of an azidyl radical from sodium azide, followed by two successive additions of this N-centered radical to the alkene, and is assisted by a Mn catalyst...
August 2018: Nature Protocols
Andrea Mariscal, Lindsay Caldarone, Jussi Tikkanen, Daisuke Nakajima, Manyin Chen, Jonathan Yeung, Marcelo Cypel, Mingyao Liu, Shaf Keshavjee
Although lung transplant is a life-saving therapy for some patients, primary graft dysfunction (PGD) is a leading cause of mortality and morbidity soon after a transplant. Ischemia reperfusion injury is known to be one of the most critical factors in PGD development. PGD is by definition an acute lung injury syndrome that occurs during the first 3 d following lung transplantation. To successfully translate laboratory discoveries to clinical practice, a reliable and practical large animal model is critical. This protocol describes a surgical technique for swine lung transplantation and postoperative management for a further 3 d post transplant...
August 2018: Nature Protocols
Yuanhui Ma, Daniel B McClatchy, Salim Barkallah, William W Wood, John R Yates
Measuring proteome response to perturbations is critical for understanding the underlying mechanisms involved. Traditional quantitative proteomic methods are limited by the large numbers of proteins in the proteome and the mass spectrometer's dynamic range. A previous method uses the biorthogonal reagent azidohomoalanine (AHA), a methionine analog, for labeling, enrichment and detection of newly synthesized proteins (NSPs). Newly synthesized AHA proteins can be coupled to biotin via CuAAC-mediated click chemistry and enriched using avidin-based affinity purification...
August 2018: Nature Protocols
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