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Immunoblotting and Immunodetection
Duojiao Ni, Peng Xu, Sean Gallagher
Immunoblotting (western blotting) is used to identify specific antigens recognized by polyclonal or monoclonal antibodies. This unit provides protocols for all steps, starting with solubilization of the protein samples, usually by means of SDS and reducing agents. Following solubilization, the
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Analysis and Purification of Mouse Intestinal Dendritic Cell and Macrophage Subsets by Flow Cytometry
Balázs Koscsó, Milena Bogunovic
The unit presents a method for analysis of intestinal dendritic cell (DC) and macrophage subsets by flow cytometry in the single cell suspension prepared from the mouse small and large intestine (Basic Protocol). describes a strategy to enrich the hematopoietic cell fraction in the sample by Percoll
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Assessment of Inflammasome Formation by Flow Cytometry
David P. Sester, Alina Zamoshnikova, Sara J. Thygesen, Parimala R. Vajjhala, Simon O. Cridland, Kate Schroder, Katryn J. Stacey
Inflammasomes are large protein complexes formed in response to cellular stresses that are platforms for recruitment and activation of caspase 1. Central to most inflammasome functions is the adapter molecule ASC (apoptosis‐associated speck‐like protein containing a caspase‐recruitment domain) that
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TepiTool: A Pipeline for Computational Prediction of T Cell Epitope Candidates
Sinu Paul, John Sidney, Alessandro Sette, Bjoern Peters
Computational prediction of T cell epitope candidates is currently being used in several applications including vaccine discovery studies, development of diagnostics, and removal of unwanted immune responses against protein therapeutics. There have been continuous improvements in the
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Burkholderia thailandensis: Growth and Laboratory Maintenance
Erin C. Garcia, Peggy A. Cotter
Burkholderia thailandensis is a nonpathogenic Gram‐negative bacterium found in tropical soils. Closely related to several human pathogens, its ease of genetic manipulation, rapid growth in the laboratory, and low virulence make B. thailandensis a commonly used model organism. This unit describes the
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Laboratory Cultivation and Maintenance of Borrelia miyamotoi
Brandee L. Stone, Catherine A. Brissette
Borrelia miyamotoi is a relapsing fever tick‐borne pathogen found in Ixodes spp. (hard) ticks. In vitro culturing has proven difficult despite initial reports of cultures maintained in Barbour‐Stoenner‐Kelly‐II (BSK‐II) medium. The ability to culture in vitro opens many avenues for investigating the
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Natural Vaccinia Virus Infection: Diagnosis, Isolation, and Characterization
Erna Geessien Kroon, Jônatas Santos Abrahão, Giliane de Souza Trindade, Graziele Pereira Oliveira, Ana Paula Moreira Franco Luiz, Galileu Barbosa Costa, Mauricio Teixeira Lima, Rafael Silva Calixto, Danilo Bretas de Oliveira, Betânia Paiva Drumond
Natural infections of Vaccinia virus (VACV)—the prototype species of the Orthopoxvirus genus, from the family Poxviridae and subfamily Chordopoxvirinae —cause an occupational emergent zoonotic disease that is primarily associated with the handling of infected dairy cattle. In humans, VACV infection
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Direct PCR of Intact Bacteria (Colony PCR)
Michael E. Woodman, Christina R. Savage, William K. Arnold, Brian Stevenson
This protocol describes an efficient method for screening intact bacteria for the presence of desired DNA sequences using the polymerase chain reaction (PCR). This method is commonly referred to as colony PCR. © 2016 by John Wiley & Sons, Inc. Keywords: colony PCR; bacteria; screen
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Use of the SHuffle Strains in Production of Proteins
Guoping Ren, Na Ke, Mehmet Berkmen
Escherichia coli continues to be a popular expression host for the production of proteins, yet successful recombinant expression of active proteins to high yields remains a trial and error process. This is mainly due to decoupling of the folding factors of a protein from its native host, when
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Separation of Peptides on HALO 2‐Micron Particles
Colin T. Mant, Robert S. Hodges
Reversed‐phase high‐performance liquid chromatography (RP‐HPLC) is of fundamental importance to the isolation and separation of peptides, proteins, and other biomolecules. Hence, there is a continuing high demand for the development of RP‐HPLC stationary‐phase materials with enhanced separation
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Introduction to Atomic Force Microscopy (AFM) in Biology
Laurent Kreplak
The atomic force microscope (AFM) has the unique capability of imaging biological samples with molecular resolution in buffer solution over a wide range of time scales from milliseconds to hours. In addition to providing topographical images of surfaces with nanometer‐ to angstrom‐scale resolution,
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Designing and Applying Proximity‐Dependent Hybridization Chain Reaction
Björn Koos, Ola Söderberg
Proximity‐dependent hybridization chain reaction (proxHCR) is a novel technique for detection of protein interaction, post‐translational modifications (PTMs), or protein expression. The method is based upon antibodies targeting the proteins of interest that are covalently conjugated to DNA
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Correlative Förster Resonance Electron Transfer‐Proximity Ligation Assay (FRET‐PLA) Technique for Studying Interactions Involving Membrane Proteins
Daniel Ivanusic, Joachim Denner, Norbert Bannert
This unit provides a guide and detailed protocol for studying membrane protein‐protein interactions (PPI) using the acceptor‐sensitized Förster resonance electron transfer (FRET) method in combination with the proximity ligation assay (PLA). The protocol in this unit is focused on the preparation of
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Rapid Preparation of a Plasma Membrane Fraction: Western Blot Detection of Translocated Glucose Transporter 4 from Plasma Membrane of Muscle and Adipose Cells and Tissues
Norio Yamamoto, Yoko Yamashita, Yasukiyo Yoshioka, Shin Nishiumi, Hitoshi Ashida
Membrane proteins account for 70% to 80% of all pharmaceutical targets, indicating their clinical relevance and underscoring the importance of identifying differentially expressed membrane proteins that reflect distinct disease properties. The translocation of proteins from the bulk of the cytosol
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Small‐Molecule‐Directed Hepatocyte‐Like Cell Differentiation of Human Pluripotent Stem Cells
Santosh Mathapati, Richard Siller, Agata A.R. Impellizzeri, Max Lycke, Karianne Vegheim, Runar Almaas, Gareth J. Sullivan
Hepatocyte‐like cells (HLCs) generated in vitro from human pluripotent stem cells (hPSCs) provide an invaluable resource for basic research, regenerative medicine, drug screening, toxicology, and modeling of liver disease and development. This unit describes a small‐molecule‐driven protocol for in
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Isolation and Assessment of Single Long‐Term Reconstituting Hematopoietic Stem Cells from Adult Mouse Bone Marrow
David G. Kent, Brad J. Dykstra, Connie J. Eaves
Hematopoietic stem cells with long‐term repopulating activity can now be routinely obtained at purities of 40% to 50% from suspensions of adult mouse bone marrow. Here we describe robust protocols for both their isolation as CD45 + EPCR + CD150 + CD48 − (ESLAM) cells using multiparameter cell
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Neural Stem Cell or Human Induced Pluripotent Stem Cell–Derived GABA‐ergic Progenitor Cell Grafting in an Animal Model of Chronic Temporal Lobe Epilepsy
Dinesh Upadhya, Bharathi Hattiangady, Geetha A. Shetty, Gabriele Zanirati, Maheedhar Kodali, Ashok K. Shetty
Grafting of neural stem cells (NSCs) or GABA‐ergic progenitor cells (GPCs) into the hippocampus could offer an alternative therapy to hippocampal resection in patients with drug‐resistant chronic epilepsy, which afflicts >30% of temporal lobe epilepsy (TLE) cases. Multipotent, self‐renewing NSCs
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Efficient Generation of Viral and Integration‐Free Human Induced Pluripotent Stem Cell‐Derived Oligodendrocytes
Araceli Espinosa‐Jeffrey, Bruno Blanchi, Juan Carlos Biancotti, Shalini Kumar, Megumi Hirose, Berhan Mandefro, Dodanim Talavera‐Adame, Nissim Benvenisty, Jean de Vellis
Here we document three highly reproducible protocols: (1) a culture system for the derivation of human oligodendrocytes (OLs) from human induced pluripotent stem cells (hiPS) and their further maturation—our protocol generates viral‐ and integration‐free OLs that efficiently commit and move forward
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Derivation of Transgene‐Free Induced Pluripotent Stem Cells from a Single Drop of Blood
Hong Yu Chen, Hong‐Kee Tan, Yuin‐Han Loh
Human‐induced pluripotent stem cells (hiPSCs) have great potential for future use in therapeutic regenerative medicine. Based on the current protocol for deriving hiPSCs, invasive procedures such as skin biopsies and venipuncture are required for obtaining donor samples. Herein, we present a
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Self‐Cloning CRISPR
Mandana Arbab, Richard I. Sherwood
CRISPR/Cas9‐gene editing has emerged as a revolutionary technology to easily modify specific genomic loci by designing complementary sgRNA sequences and introducing these into cells along with Cas9. Self‐cloning CRISPR/Cas9 (scCRISPR) uses a self‐cleaving palindromic sgRNA plasmid (sgPal) that
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Comprehensive Protocols for CRISPR/Cas9‐based Gene Editing in Human Pluripotent Stem Cells
David P. Santos, Evangelos Kiskinis, Kevin Eggan, Florian T. Merkle
Genome editing of human pluripotent stem cells (hPSCs) with the CRISPR/Cas9 system has the potential to revolutionize hPSC‐based disease modeling, drug screening, and transplantation therapy. Here, we aim to provide a single resource to enable groups, even those with limited experience with hPSC
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A High‐Throughput Screening Assay to Identify Kidney Toxic Compounds
Susanne Ramm, Melanie Adler, Vishal S. Vaidya
Kidney toxicity due to drugs and chemicals poses a significant health burden for patients and a financial risk for pharmaceutical companies. However, currently no sensitive and high‐throughput in vitro method exists for predictive nephrotoxicity assessment. Primary human proximal tubular epithelial
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Methods for the Detection of Autophagy in Mammalian Cells
Ziyan Zhang, Rajat Singh, Michael Aschner
Macroautophagy (hereafter referred to as autophagy) is a degradation pathway that delivers cytoplasmic materials to lysosomes via double‐membraned vesicles designated autophagosomes. Cytoplasmic constituents are sequestered into autophagosomes, which subsequently fuse with lysosomes, where the cargo
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In Vivo Determination of Mitochondrial Function Using Luciferase‐Expressing Caenorhabditis elegans: Contribution of Oxidative Phosphorylation, Glycolysis, and Fatty Acid Oxidation to Toxicant‐Induced Dysfunction
Anthony L. Luz, Cristina Lagido, Matthew D. Hirschey, Joel N. Meyer
Mitochondria are a target of many drugs and environmental toxicants; however, how toxicant‐induced mitochondrial dysfunction contributes to the progression of human disease remains poorly understood. To address this issue, in vivo assays capable of rapidly assessing mitochondrial function need to be
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