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Phos-tag Immunoblot Analysis for Detecting IRF5 Phosphorylation

Featured protocol,  Authors: Go R. Sato
Go R. SatoAffiliation: Department of Immunology, Yokohama City University Graduate School of Medicine, Yokohama, Japan
Bio-protocol author page: a4548
Tatsuma Ban
Tatsuma BanAffiliation: Department of Immunology, Yokohama City University Graduate School of Medicine, Yokohama, Japan
For correspondence: tatban@yokohama-cu.ac.jp
Bio-protocol author page: a4549
 and Tomohiko Tamura
Tomohiko TamuraAffiliation: Department of Immunology, Yokohama City University Graduate School of Medicine, Yokohama, Japan
For correspondence: tamurat@yokohama-cu.ac.jp
Bio-protocol author page: a4550
date: 5/20/2017, 126 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2295.

Brief version appeared in Immunity, Aug 2016
While the activation of the transcription factor interferon regulatory factor 5 (IRF5) is critical for the induction of innate immune responses, it also contributes to the pathogenesis of the autoimmune disease systemic lupus erythematosus (SLE). IRF5 phosphorylation is a hallmark of its activation in the Toll-like receptor (TLR) pathway, where active IRF5 induces type I interferon and proinflammatory cytokine genes. By using the phosphate-binding molecule Phos-tag, without either radioisotopes or phospho-specific antibodies, the protocol described here enables detection of the phosphorylation of both human and murine IRF5, as well as that of other proteins.

Assaying the Effects of Splice Site Variants by Exon Trapping in a Mammalian Cell Line

Featured protocol,  Authors: Stuart W. Tompson
Stuart W. TompsonAffiliation: Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison, Madison, Wisconsin, USA
For correspondence: stompson@wisc.edu
Bio-protocol author page: a4509
 and Terri L. Young
Terri L. YoungAffiliation: Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison, Madison, Wisconsin, USA
Bio-protocol author page: a4510
date: 5/20/2017, 98 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2281.

Brief version appeared in J Clin Invest, Jul 2016
There are several in silico programs that endeavor to predict the functional impact of an individual’s sequence variation at splice donor/acceptor sites, but experimental confirmation is problematic without a source of RNA from the individual that carries the variant. With the aid of an exon trapping vector, such as pSPL3, an investigator can test whether a splice site sequence change leads to altered RNA splicing, through expression of reference and variant mini-genes in mammalian cells and analysis of the resultant RNA products.

Spore Preparation Protocol for Enrichment of Clostridia from Murine Intestine

Featured protocol,  Authors: Eric M. Velazquez
Eric M. VelazquezAffiliation: Department of Medical Microbiology and Immunology, School of Medicine, University of California at Davis, One Shields Avenue, Davis, CA 95616, USA
Bio-protocol author page: a4551
Fabian Rivera-Chávez
Fabian Rivera-ChávezAffiliation: Department of Medical Microbiology and Immunology, School of Medicine, University of California at Davis, One Shields Avenue, Davis, CA 95616, USA
Bio-protocol author page: a4552
 and Andreas J. Bäumler
Andreas J. BäumlerAffiliation: Department of Medical Microbiology and Immunology, School of Medicine, University of California at Davis, One Shields Avenue, Davis, CA 95616, USA
For correspondence: ajbaumler@ucdavis.edu
Bio-protocol author page: a4553
date: 5/20/2017, 168 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2296.

Brief version appeared in Cell Host Microbe, Apr 2016
In recent years, many spore-forming commensal Clostridia found in the gut have been discovered to promote host physiology, immune development, and protection against infections. We provide a detailed protocol for rapid enrichment of spore-forming bacteria from murine intestine. Briefly, contents from the intestinal cecum are collected aerobically, diluted and finally treated with chloroform to enrich for Clostridia spores.

Creating a RAW264.7 CRISPR-Cas9 Genome Wide Library

Featured protocol,  Authors: Brooke A Napier
Brooke A Napier Affiliation: Department of Microbiology and Immunology, Stanford School of Medicine, Stanford University, Stanford, CA, USA
Bio-protocol author page: a4566
 and Denise M Monack
Denise M MonackAffiliation: Department of Microbiology and Immunology, Stanford School of Medicine, Stanford University, Stanford, CA, USA
For correspondence: dmonack@stanford.edu
Bio-protocol author page: a4567
date: 5/20/2017, 112 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2320.

Brief version appeared in J Exp Med, Oct 2016
The bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 genome editing tools are used in mammalian cells to knock-out specific genes of interest to elucidate gene function. The CRISPR-Cas9 system requires that the mammalian cell expresses Cas9 endonuclease, guide RNA (gRNA) to lead the endonuclease to the gene of interest, and the PAM sequence that links the Cas9 to the gRNA. CRISPR-Cas9 genome wide libraries are used to screen the effect of each gene in the genome on the cellular phenotype of interest, in an unbiased high-throughput manner. In this protocol, we describe our method of creating a CRISPR-Cas9 genome wide library in a transformed murine macrophage cell-line (RAW264.7). We have employed this library to identify novel mediators in the caspase-11 cell death pathway (Napier et al., 2016); however, this library can then be used to screen the importance of specific genes in multiple murine macrophage cellular pathways.

Detection of ASC Oligomerization by Western Blotting

Featured protocol,  Authors: Jérôme Lugrin
Jérôme LugrinAffiliation: Department of Biochemistry, University of Lausanne, CH-1066 Epalinges, Switzerland
Bio-protocol author page: a4538
 and Fabio Martinon
Fabio Martinon Affiliation: Department of Biochemistry, University of Lausanne, CH-1066 Epalinges, Switzerland
For correspondence: Fabio.Martinon@unil.ch
Bio-protocol author page: a4539
date: 5/20/2017, 95 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2292.

Brief version appeared in Proc Natl Acad Sci U S A, Aug 2016
The apoptosis-associated speck-like protein with a caspase-recruitment domain (ASC) adaptor protein bridges inflammasome sensors and caspase-1. Upon inflammasome activation, ASC nucleates in a prion-like manner into a large and single platform responsible for the recruitment and the activation of caspase-1. Active caspase-1 will in turn promote the proteolytic maturation of the pro-inflammatory cytokine IL-1β. ASC oligomerization is a direct evidence for inflammasome activation and its detection allows a read-out independent of caspase-1 and IL-1β. This protocol describes how to detect the oligomerization of ASC by Western blot.

Muscle Histology Characterization Using H&E Staining and Muscle Fiber Type Classification Using Immunofluorescence Staining

Featured protocol,  Authors: Chao Wang
Chao WangAffiliation: Department of Animal Science, Purdue University, West Lafayette, Indiana, USA
For correspondence: wang1438@purdue.edu
Bio-protocol author page: a4505
Feng Yue
Feng YueAffiliation: Department of Animal Science, Purdue University, West Lafayette, Indiana, USA
Bio-protocol author page: a4506
 and Shihuan Kuang
Shihuan KuangAffiliation 1: Department of Animal Science, Purdue University, West Lafayette, Indiana, USA
Affiliation 2: Center for Cancer Research, Purdue University, West Lafayette, Indiana, USA
For correspondence: skuang@purdue.edu
Bio-protocol author page: a2572
date: 5/20/2017, 111 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2279.

Brief version appeared in Elife, Sep 2016
Muscle function is determined by its structure and fiber type composition. Here we describe a protocol to examine muscle histology and myofiber types using hematoxylin and eosin (H&E) and immunofluorescence staining, respectively. H&E stain nucleus in blue and cytoplasm in red, therefore allowing for morphological analyses, such as myofiber diameter, the presence of degenerated and regenerated myofibers, and adipocytes and fibrotic cells. Muscle fibers in adult skeletal muscles of rodents are classified into 4 subtypes based on the expression of myosin heavy chain proteins: Myh7 (type I fiber), Myh2 (type IIA fiber), Myh1 (type IIX fiber), Myh4 (type IIB fiber). A panel of monoclonal antibodies can be used to specifically label these muscle fiber subtypes. These protocols are commonly used in the study of muscle development, growth and regeneration (for example: Wang et al., 2015; Nie et al., 2016; Yue et al., 2016; Wang et al., 2017).

Isolation of Murine Alveolar Type II Epithelial Cells

Featured protocol,  Authors: Fan Sun
Fan SunAffiliation 1: University of Pittsburgh Cancer Institute, University of Pittsburgh, Pittsburgh, USA
Affiliation 2: Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, USA
Bio-protocol author page: a4527
Gutian Xiao
Gutian XiaoAffiliation 1: University of Pittsburgh Cancer Institute, University of Pittsburgh, Pittsburgh, USA
Affiliation 2: Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, USA
Bio-protocol author page: a4528
 and Zhaoxia Qu
Zhaoxia QuAffiliation 1: University of Pittsburgh Cancer Institute, University of Pittsburgh, Pittsburgh, USA
Affiliation 2: Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, USA
For correspondence: quz@upmc.edu
Bio-protocol author page: a4529
date: 5/20/2017, 125 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2288.

Brief version appeared in Oncogene, May 2016
We have optimized a protocol for isolation of alveolar type II epithelial cells from mouse lung. Lung cell suspensions are prepared by intratracheal instillation of dispase and agarose followed by mechanical disaggregation of the lungs. Alveolar type II epithelial cells are purified from these lung cell suspensions through magnetic-based negative selection using a Biotin-antibody, Streptavidin-MicroBeads system. The purified alveolar type II epithelial cells can be cultured and maintained on fibronectin-coated plates in DMEM with 10% FBS. This protocol enables specific investigation of alveolar type II epithelial cells at molecular and cellular levels and provides an important tool to investigate in vitro the mechanisms underlying lung pathogenesis.

Murine Bronchoalveolar Lavage

Featured protocol,  Authors: Fan Sun
Fan SunAffiliation 1: University of Pittsburgh Cancer Institute, University of Pittsburgh, Pittsburgh, USA
Affiliation 2: Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, USA
Bio-protocol author page: a4527
Gutian Xiao
Gutian XiaoAffiliation 1: University of Pittsburgh Cancer Institute, University of Pittsburgh, Pittsburgh, USA
Affiliation 2: Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, USA
Bio-protocol author page: a4528
 and Zhaoxia Qu
Zhaoxia QuAffiliation 1: University of Pittsburgh Cancer Institute, University of Pittsburgh, Pittsburgh, USA
Affiliation 2: Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, USA
For correspondence: quz@upmc.edu
Bio-protocol author page: a4529
date: 5/20/2017, 117 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2287.

Brief version appeared in Oncogene, May 2016
A basic Bronchoalveolar lavage (BAL) procedure in mouse is described here. Cells and fluids obtained from BAL can be analyzed by Hema3-staining, immunostaining, Fluorescence-activated cell sorting (FACS), PCR, bicinchoninic acid protein assay, enzyme-linked immunosorbent assay (ELISA), luminex assays, etc., to examine the immune cells, pathogens, proteins such as cytokines/chemokines, and the expression levels of inflammation-related and other genes in the cells. This will help to understand the underlying mechanisms of these lung diseases and develop specific and effective drugs.

Analysis of in vivo Interaction between RNA Binding Proteins and Their RNA Targets by UV Cross-linking and Immunoprecipitation (CLIP) Method

Featured protocol,  Authors: Pamela Bielli
Pamela BielliAffiliation 1: Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy
Affiliation 2: Laboratory of Neuroembriology, Fondazione Santa Lucia, Rome, Italy
Bio-protocol author page: a4492
 and Claudio Sette
Claudio SetteAffiliation 1: Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy
Affiliation 2: Laboratory of Neuroembriology, Fondazione Santa Lucia, Rome, Italy
For correspondence: claudio.sette@uniroma2.it
Bio-protocol author page: a4493
date: 5/20/2017, 152 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2274.

Brief version appeared in Oncogene, Apr 2016
RNA metabolism is tightly controlled across different tissues and developmental stages, and its dysregulation is one of the molecular hallmarks of cancer. Through direct binding to specific sequence element(s), RNA binding proteins (RBPs) play a pivotal role in co- and post-transcriptional RNA regulatory events. We have recently demonstrated that, in pancreatic cancer cells, acquisition of a drug resistant (DR)-phenotype relied on upregulation of the polypyrimidine tract binding protein (PTBP1), which in turn is recruited to the pyruvate kinase pre-mRNA and favors splicing of the oncogenic PKM2 variant. Herein, we describe a step-by-step protocol of the ultraviolet (UV) light cross-linking and immunoprecipitation (CLIP) method to determine the direct binding of a RBP to specific regions of its target RNAs in adherent human cell lines.

Nucleosome Positioning Assay

Featured protocol,  Authors: Zhongliang Zhao
Zhongliang ZhaoAffiliation: Division of Molecular Biology of the Cell II, German Cancer Research Center, DKFZ-ZMBH Alliance, Im Neuenheimer Feld 581, 69120 Heidelberg, Germany
Bio-protocol author page: a4525
 and Holger Bierhoff
Holger BierhoffAffiliation 1: Department of Biochemistry, Institute of Biochemistry and Biophysics, Center for Molecular Biomedicine (CMB), Friedrich Schiller University Jena, Hans-Knöll-Str. 2, Jena, Germany
Affiliation 2: Leibniz-Institute on Aging – Fritz Lipmann Institute (FLI), Beutenbergstrasse 11, Jena, Germany
For correspondence: holger.bierhoff@uni-jena.de
Bio-protocol author page: a4526
date: 5/20/2017, 102 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2285.

Brief version appeared in Cell Rep, Mar 2016
The basic unit of chromatin is the nucleosome, a histone octamer with 147 base pairs of DNA wrapped around it. Positions of nucleosomes relative to each other and to DNA elements have a strong impact on chromatin structure and gene activity and are tightly regulated at multiple levels, i.e., DNA sequence, transcription factor binding, histone modifications and variants, and chromatin remodeling enzymes (Bell et al., 2011; Hughes and Rando, 2014). Nucleosome positions in cells or isolated nuclei can be detected by partial nuclease digestion of native or cross-linked chromatin followed by ligation-mediated polymerase chain reaction (LM-PCR) (McPherson et al., 1993; Soutoglou and Talianidis, 2002). This protocol describes a nucleosome positioning assay using Micrococcal Nuclease (MNase) digestion of formaldehyde-fixed chromatin followed by LM-PCR. We exemplify the nucleosome positioning assay for the promoter of genes encoding ribosomal RNA (rRNA genes or rDNA) in mice, which has two mutually exclusive configurations. The rDNA promoter harbors either an upstream nucleosome (NucU) covering nucleotides -157 to -2 relative to the transcription start site, or a downstream nucleosome (NucD) at position -132 to +22 (Li et al., 2006; Xie et al., 2012). Radioactive labeling of LM-PCR products followed by denaturing urea-polyacrylamide gel electrophoresis allows resolution and relative quantification of both configurations. As depicted in the diagram in Figure 1, the nucleosome positioning assay is a versatile low to medium throughput method to map discrete nucleosome positions with high precision in a semi-quantitative manner.

Lung Section Staining and Microscopy

Featured protocol,  Authors: Xiaofeng Zhou
Xiaofeng ZhouAffiliation: Department of Internal Medicine, Pulmonary and Critical Care Medicine Division, University of Michigan, Ann Arbor, Michigan, USA
For correspondence: xiazhou@umich.edu
Bio-protocol author page: a4212
 and Bethany B Moore
Bethany B MooreAffiliation: Department of Internal Medicine, Pulmonary and Critical Care Medicine Division, University of Michigan, Ann Arbor, Michigan, USA
Bio-protocol author page: a4213
date: 5/20/2017, 107 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2286.

Brief version appeared in Mucosal Immunol, May 2016
Our protocol describes immunofluorescent staining, hematoxylin and eosin staining and Masson’s trichrome staining on lung sections.

Isolation and Cultivation of Primary Brain Endothelial Cells from Adult Mice

Featured protocol,  Authors: Julian Christopher Assmann
Julian Christopher AssmannAffiliation: Institute of Experimental and Clinical Pharmacology and Toxicology, University of Lübeck, Lübeck, Germany
Bio-protocol author page: a4540
Kristin Müller
Kristin MüllerAffiliation: Institute of Experimental and Clinical Pharmacology and Toxicology, University of Lübeck, Lübeck, Germany
Bio-protocol author page: a4541
Jan Wenzel
Jan WenzelAffiliation: Institute of Experimental and Clinical Pharmacology and Toxicology, University of Lübeck, Lübeck, Germany
Bio-protocol author page: a4542
Thomas Walther
Thomas WaltherAffiliation: Institute of Experimental and Clinical Pharmacology and Toxicology, University of Lübeck, Lübeck, Germany
Bio-protocol author page: a4543
Josefine Brands
Josefine BrandsAffiliation: Institute of Experimental and Clinical Pharmacology and Toxicology, University of Lübeck, Lübeck, Germany
Bio-protocol author page: a4544
Peter Thornton
Peter ThorntonAffiliation: Innovative Medicines and Early Development, Neuroscience, AstraZeneca, Cambridge, United Kingdom
Bio-protocol author page: a4545
Stuart M. Allan
Stuart M. AllanAffiliation: Division of Neuroscience & Experimental Psychology, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, United Kingdom
Bio-protocol author page: a4546
 and Markus Schwaninger
Markus SchwaningerAffiliation: Institute of Experimental and Clinical Pharmacology and Toxicology, University of Lübeck, Lübeck, Germany
For correspondence: markus.schwaninger@pharma.uni-luebeck.de
Bio-protocol author page: a4547
date: 5/20/2017, 136 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2294.

Brief version appeared in J Neurosci, Sep 2016
Brain endothelial cells are the major building block of the blood-brain barrier. To study the role of brain endothelial cells in vitro, the isolation of primary cells is of critical value. Here, we describe a protocol in which vessel fragments are isolated from adult mice. After density centrifugation and mild digestion of the fragments, outgrowing endothelial cells are selected by puromycin treatment and grown to confluence within one week.

A Tactile-visual Conditional Discrimination Task for Testing Spatial Working Memory in Rats

Featured protocol,  Authors: Alicia Edsall
Alicia EdsallAffiliation: Department of Psychological and Brain Sciences, University of Delaware, Delaware, USA
Bio-protocol author page: a4511
Zachary Gemzik
Zachary GemzikAffiliation: Department of Psychological and Brain Sciences, University of Delaware, Delaware, USA
Bio-protocol author page: a4512
 and Amy Griffin
Amy GriffinAffiliation: Department of Psychological and Brain Sciences, University of Delaware, Delaware, USA
For correspondence: amygriff@psych.udel.edu
Bio-protocol author page: a4513
date: 5/20/2017, 115 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2282.

Brief version appeared in J Neurosci, Aug 2016
This protocol describes a novel dual task comparison across two variants of a tactile-visual conditional discrimination (CD) T-maze task, one is dependent upon spatial working memory (SWM; CDWM) and the other one (CDSTANDARD) is not. The task variants are equivalent in their sensory and motor requirements and overt behavior of the rat. Therefore, differences between the two task variants in the dependent variables such as choice accuracy, neural firing patterns, and the effects of pharmacological or optogenetic inactivation in brain regions of interest can be attributed to SWM, ruling out confounding sensorimotor variables, such as tactile, visual and self-motion cues. The CDWM task protocol is published in Hallock et al., 2013b and Urban et al., 2014.

Efficient Production of Functional Human NKT Cells from Induced Pluripotent Stem Cells − Reprogramming of Human Vα24+iNKT Cells

Featured protocol,  Authors: Daisuke Yamada
Daisuke YamadaAffiliation: Laboratory for Developmental Genetics, Center for Integrative Medical Sciences (IMS), RIKEN, Yokohama, Japan
For correspondence: daisuke.yamada@riken.jp
Bio-protocol author page: a4503
Tomonori Iyoda
Tomonori IyodaAffiliation: Laboratory for Immunotherapy, Center for Integrative Medical Sciences (IMS), RIKEN, Yokohama, Japan
Bio-protocol author page: a4497
Kanako Shimizu
Kanako ShimizuAffiliation: Laboratory for Immunotherapy, Center for Integrative Medical Sciences (IMS), RIKEN, Yokohama, Japan
Bio-protocol author page: a4498
Yusuke Sato
Yusuke SatoAffiliation: Laboratory for Immunotherapy, Center for Integrative Medical Sciences (IMS), RIKEN, Yokohama, Japan
Bio-protocol author page: a4501
Haruhiko Koseki
Haruhiko KosekiAffiliation: Laboratory for Developmental Genetics, Center for Integrative Medical Sciences (IMS), RIKEN, Yokohama, Japan
Bio-protocol author page: a4502
 and Shin-ichiro Fujii
Shin-ichiro FujiiAffiliation: Laboratory for Immunotherapy, Center for Integrative Medical Sciences (IMS), RIKEN, Yokohama, Japan
For correspondence: shin-ichiro.fujii@riken.jp
Bio-protocol author page: a4504
date: 5/20/2017, 93 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2277.

Brief version appeared in Stem Cells, Dec 2016
Antigen-specific T cell-derived induced pluripotent stem cells (iPSCs) have been shown to re-differentiate into functional T cells and thus provide a potential source of T cells that could be useful for cancer immunotherapy. Human Vα24+ invariant natural killer T (Vα24+iNKT) cells are subset of T cells that are characterized by the expression of an invariant Vα24-Jα18 paired with Vβ11, that recognize glycolipids, such as α-galactosylceramide (α-GalCer), presented by the MHC class I-like molecule CD1d. Vα24+iNKT cells capable of producing IFN-γ are reported to augment anti-tumor responses, which affects both NK cells and CD8+ cytotoxic T lymphocytes to eliminate MHC- and MHC+ tumor cells, respectively. Here we describe a robust protocol to reprogram human Vα24+iNKT cells into iPSC, and then to re-differentiate them into Vα24+iNKT cells (iPS-Vα24+iNKT). We further provide a protocol to measure the activity of iPS-Vα24+iNKT cells.

Primary Olfactory Ensheathing Cell Culture from Human Olfactory Mucosa Specimen

Featured protocol,  Authors: Mansoureh Hashemi
Mansoureh HashemiAffiliation: Functional neurosurgery research center, Shahid Beheshti University of medical sciences, Tehran, Iran
For correspondence: mansooreh.hashemi@yahoo.com
Bio-protocol author page: a4391
 and Mahmoudreza Hadjighassem
Mahmoudreza HadjighassemAffiliation 1: Department of Neuroscience, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran
Affiliation 2: Brain and Spinal Cord Injury Research Center, Neuroscience Institute, Tehran University of Medical Sciences, Tehran, Iran
Bio-protocol author page: a4392
date: 5/20/2017, 100 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2275.

Brief version appeared in Mol Neurobiol, Oct 2016
The human olfactory mucosa is located in the middle and superior turbinates, and the septum of nasal cavity. Olfactory mucosa plays an important role in detection of odours and it is also the only nervous tissue that is exposed to the external environment. This property leads to easy access to the olfactory mucosa for achieving various researches. The lamina propria of olfactory mucosa consists of olfactory ensheathing cells (OECs) that cover the nerve fibers of olfactory. Here we describe a protocol for isolation of OECs from biopsy of human olfactory mucosa.

Flow Cytometric Analysis of Drug-induced HIV-1 Transcriptional Activity in A2 and A72 J-Lat Cell Lines

Featured protocol,  Authors: Daniela Boehm
Daniela BoehmAffiliation: Gladstone Institute of Virology and Immunology, San Francisco, CA, USA
Bio-protocol author page: a4533
 and Melanie Ott
Melanie OttAffiliation 1: Gladstone Institute of Virology and Immunology, San Francisco, CA, USA
Affiliation 2: Department of Medicine, University of California, San Francisco, CA, USA
For correspondence: mott@gladstone.ucsf.edu
Bio-protocol author page: a2100
date: 5/20/2017, 108 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2290.

Brief version appeared in Cell Cycle, Feb 2013
The main obstacle to eradicating HIV-1 from patients is post-integration latency (Finzi et al., 1999). Antiretroviral treatments target only actively replicating virus, while latent infections that have low or no transcriptional activity remain untreated (Sedaghat et al., 2007). A combination of antiretroviral treatments with latency-purging strategies may accelerate the depletion of latent reservoirs and lead to a cure (Geeraert et al., 2008). Current strategies to reactivate HIV-1 from latency include use of prostratin, a non-tumor-promoting phorbol ester (Williams et al., 2004), BET inhibitors (Filippakopoulos et al., 2010; Delmore et al., 2011), and histone deacetylase (HDAC) inhibitors, such as suberoylanilidehydroxamic acid (i.e., SAHA or Vorinostat) (Kelly et al., 2003; Archin et al., 2009; Contreras et al., 2009; Edelstein et al., 2009). As the mechanisms of HIV-1 latency are diverse, effective reactivation may require combinatorial strategies (Quivy et al., 2002). The following protocol describes a flow cytometry-based method to quantify transcriptional activation of the HIV-1 long terminal repeat (LTR) upon drug treatment. This protocol is optimized for studying latently HIV-1-infected Jurkat (J-Lat) cell lines that contain a GFP cassette. J-Lats that contain a different reporter, for example Luciferase, can be treated with drugs as described but have to be analyzed differently.

Semi-quantitative Analysis of H4K20me1 Levels in Living Cells Using Mintbody

Featured protocol,  Authors: Yuko Sato
Yuko SatoAffiliation: Cell Biology Unit, Institute of Innovative Research, Tokyo Institute of Technology, Yokohama, Japan
For correspondence: satoy@bio.titech.ac.jp
Bio-protocol author page: a4494
 and Hiroshi Kimura
Hiroshi KimuraAffiliation: Cell Biology Unit, Institute of Innovative Research, Tokyo Institute of Technology, Yokohama, Japan
For correspondence: hkimura@bio.titech.ac.jp
Bio-protocol author page: a4495
date: 5/20/2017, 138 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2276.

Brief version appeared in J Mol Biol, Oct 2016
Eukaryotic nuclear DNA wraps around histone proteins to form a nucleosome, a basic unit of chromatin. Posttranslational modification of histones play an important role in gene regulation and chromosome duplication. Some modifications are quite stable to be an epigenetic memory, and others exhibit rapid turnover or fluctuate during the cell cycle. Histone H4 Lys20 monomethylation (H4K20me1) has been shown to be involved in chromosome condensation, segregation, replication and repair. H4K20 methylation is controlled through a few methyltransferases, PR-Set7/Set8, SUV420H1, and SUV420H2, and a demethylase, PHF8. In cycling cells, the level of H4K20me1 increases during G2 and M phases and decreases during G1 phase. To monitor the local concentration and global fluctuation of histone modifications in living cells, we have developed a genetically encoded probe termed mintbody (modification-specific intracellular antibody; Sato et al., 2013 and 2016). By measuring the nuclear to cytoplasmic intensity ratio, the relative level of H4K20me1 in individual cells can be monitored. This detailed protocol allows the semi-quantitative analysis of the effects of methyltransferases on H4K20me1 levels in living cells based on H4K20me1-mintbody described by Sato et al. (2016).

A Method to Convert mRNA into a Guide RNA (gRNA) Library without Requiring Previous Bioinformatics Knowledge of the Organism

Featured protocol,  Author: Hiroshi Arakawa
Hiroshi ArakawaAffiliation: IFOM–FIRC Institute of Molecular Oncology Foundation, Milan, Italy
For correspondence: hiroshi.arakawa@ifom.eu
Bio-protocol author page: a4565
date: 5/20/2017, 93 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2319.

Brief version appeared in Sci Adv, Aug 2016
While the diversity of species represents a diversity of special biological abilities, many of the genes that encode those special abilities in a variety of species are untouched, leaving an untapped gold mine of genetic information; however, despite current advances in genome bioinformatics, annotation of that genetic information is incomplete in most species, except for well-established model organisms, such as human, mouse, or yeast. A guide RNA (gRNA) library using the clustered regularly interspersed palindromic repeats (CRISPR)/Cas9 (CRISPR-associated protein 9) system can be used for the phenotypic screening of uncharacterized genes by forward genetics. The construction of a gRNA library usually requires an abundance of chemically synthesized oligos designed from annotated genes; if one wants to convert mRNA into gRNA without prior knowledge of the target DNA sequences, the major challenges are finding the sequences flanking the protospacer adjacent motif (PAM) and cutting out the 20-bp fragment. Recently, I developed a molecular biology-based technique to convert mRNA into a gRNA library (Arakawa, 2016) (Figure 1). Here I describe the detailed protocol of how to construct a gRNA library from mRNA.

Peripheral Nerve Injury: a Mouse Model of Neuropathic Pain

Featured protocol,  Authors: Takahiro Masuda
Takahiro MasudaAffiliation: Institute of Neuropathology, University of Freiburg, Freiburg, Germany
Bio-protocol author page: a4442
Yuta Kohro
Yuta KohroAffiliation: Department of Life Innovation, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan
Bio-protocol author page: a4443
Kazuhide Inoue
Kazuhide InoueAffiliation: Department of Molecular and System Pharmacology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan
Bio-protocol author page: a4444
 and Makoto Tsuda
Makoto TsudaAffiliation: Department of Life Innovation, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan
For correspondence: tsuda@phar.kyushu-u.ac.jp
Bio-protocol author page: a4445
date: 5/5/2017, 201 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2252.

Brief version appeared in Nat Commun, Aug 2016
Neuropathic pain is one of the highly debilitating chronic pain conditions, for which, currently, there is no therapeutic treatment. In order to reveal the underlying mechanism for neuropathic pain, various animal models have been established (Burma et al., 2016). This protocol describes how to prepare spinal nerve injury model (Kim and Chung, 1992; Rigaud et al., 2008; Masuda et al., 2016), one of the most frequently-used and highly reproducible models in which multiple alterations occur both in the peripheral and central nervous system.

Isolation, Culturing, and Differentiation of Primary Myoblasts from Skeletal Muscle of Adult Mice

Featured protocol,  Authors: Lubna Hindi
Lubna HindiAffiliation: Department of Anatomical Sciences and Neurobiology, University of Louisville School of Medicine, Louisville, USA
Bio-protocol author page: a4417
Joseph D. McMillan
Joseph D. McMillanAffiliation: Department of Anatomical Sciences and Neurobiology, University of Louisville School of Medicine, Louisville, USA
Bio-protocol author page: a4418
Dil Afroze
Dil AfrozeAffiliation: Department of Anatomical Sciences and Neurobiology, University of Louisville School of Medicine, Louisville, USA
Bio-protocol author page: a4419
Sajedah M. Hindi
Sajedah M. HindiAffiliation: Department of Anatomical Sciences and Neurobiology, University of Louisville School of Medicine, Louisville, USA
Bio-protocol author page: a3536
 and Ashok Kumar
Ashok KumarAffiliation 1: Department of Anatomical Sciences and Neurobiology, University of Louisville School of Medicine, Louisville, USA
Affiliation 2: Professor and Distinguished University Scholar, Department of Anatomical Sciences and Neurobiology, University of Louisville School of Medicine, Louisville, USA
For correspondence: ashok.kumar@louisville.edu
Bio-protocol author page: a3537
date: 5/5/2017, 243 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2248.

Brief version appeared in Nat Commun, Dec 2015
Myogenesis is a multi-step process that leads to the formation of skeletal muscle during embryonic development and repair of injured myofibers. In this process, myoblasts are the main effector cell type which fuse with each other or to injured myofibers leading to the formation of new myofibers or regeneration of skeletal muscle in adults. Many steps of myogenesis can be recapitulated through in vitro differentiation of myoblasts into myotubes. Most laboratories use immortalized myogenic cells lines that also differentiate into myotubes. Although these cell lines have been found quite useful to delineating the regulatory mechanisms of myogenesis, they often show a great degree of variability depending on the origin of the cells and culture conditions. Primary myoblasts have been suggested as the most physiologically relevant model for studying myogenesis in vitro. However, due to their low abundance in adult skeletal muscle, isolation of primary myoblasts is technically challenging. In this article, we describe an improved protocol for the isolation of primary myoblasts from adult skeletal muscle of mice. We also describe methods for their culturing and differentiation into myotubes.

Immunostaining of Formaldehyde-fixed Metaphase Chromosome from Untreated and Aphidicolin-treated DT40 Cells

Featured protocol,  Author: Vibe H. Oestergaard
Vibe H. OestergaardAffiliation: Department of Biology, University of Copenhagen, Ole Maaloees Vej 5, Copenhagen N, Denmark
For correspondence: vibe@bio.ku.dk
Bio-protocol author page: a4458
date: 5/5/2017, 175 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2259.

Brief version appeared in J Cell Biol, Aug 2015
During mitosis chromosomes are condensed into dense X-shaped structures that allow for microscopic determination of karyotype as well as inspection of chromosome morphology.

Relative Stiffness Measurements of Tumour Tissues by Shear Rheology

Featured protocol,  Authors: Chris D. Madsen
Chris D. MadsenAffiliation: Department of Laboratory Medicine, Division of Translational Cancer Research, Lund University, Lund, Sweden
For correspondence: chris.madsen@med.lu.se
Bio-protocol author page: a3987
 and Thomas R. Cox
Thomas R. CoxAffiliation: The Garvan Institute of Medical Research and The Kinghorn Cancer Centre, Cancer Division, St Vincent's Clinical School, Faculty of Medicine, UNSW Sydney, Australia
For correspondence: t.cox@garvan.org.au
Bio-protocol author page: a3986
date: 5/5/2017, 236 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2265.

Brief version appeared in EMBO Rep, Oct 2015
The microenvironment of solid tumours is a critical contributor to the progression of tumours and offers a promising target for therapeutic intervention (Cox and Erler, 2011; Barker et al., 2012; Cox et al., 2016; Cox and Erler, 2016). The properties of the tumour microenvironment vary significantly from that of the original tissue in both biochemistry and biomechanics. At present, the complex interplay between the biomechanical properties of the microenvironment and tumour cell phenotype are under intense investigation. The ability to measure the biomechanical properties of tumour samples from cancer models will increase our understanding of their importance in solid tumour biology. Here we report a simple method to measure the viscoelastic properties of tumour specimens using a controlled strain rotational rheometer.

Olfactory Cued Learning Paradigm

Featured protocol,  Authors: Gary Liu
Gary LiuAffiliation 1: Program in Developmental Biology, Baylor College of Medicine, Houston, USA
Affiliation 2: Medical Scientist Training Program, Baylor College of Medicine, Houston, USA
For correspondence: garyliu87@gmail.com
Bio-protocol author page: a4435
Cynthia K. McClard*
Cynthia K. McClardAffiliation 1: Medical Scientist Training Program, Baylor College of Medicine, Houston, USA
Affiliation 2: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, USA
Bio-protocol author page: a4436
Burak Tepe*
Burak TepeAffiliation: Program in Developmental Biology, Baylor College of Medicine, Houston, USA
Bio-protocol author page: a4437
Jessica Swanson
Jessica SwansonAffiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, USA
Bio-protocol author page: a4438
Brandon Pekarek
Brandon PekarekAffiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, USA
Bio-protocol author page: a4439
Sugi Panneerselvam
Sugi PanneerselvamAffiliation: Rice University, Houston, USA
Bio-protocol author page: a4440
 and Benjamin R. Arenkiel
Benjamin R. ArenkielAffiliation 1: Program in Developmental Biology, Baylor College of Medicine, Houston, USA
Affiliation 2: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, USA
Affiliation 3: Department of Neuroscience, Baylor College of Medicine, Houston, USA
Affiliation 4: Jan and Dan Duncan Neurological Research Institute at Texas Children’s Hospital, Houston, USA
Bio-protocol author page: a4441
 (*contributed equally to this work) date: 5/5/2017, 151 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2251.

Brief version appeared in J Neurosci, Aug 2016
Sensory stimulation leads to structural changes within the CNS (Central Nervous System), thus providing the fundamental mechanism for learning and memory. The olfactory circuit offers a unique model for studying experience-dependent plasticity, partly due to a continuous supply of integrating adult born neurons. Our lab has recently implemented an olfactory cued learning paradigm in which specific odor pairs are coupled to either a reward or punishment to study downstream circuit changes. The following protocol outlines the basic set up for our learning paradigm. Here, we describe the equipment setup, programming of software, and method of behavioral training.

Analysis of Mitochondrial Transfer in Direct Co-cultures of Human Monocyte-derived Macrophages (MDM) and Mesenchymal Stem Cells (MSC)

Featured protocol,  Authors: Megan V. Jackson
Megan V. JacksonAffiliation: Centre for Experimental Medicine, School of Medicine, Dentistry and Biomedical Sciences, Queen’s University Belfast, Belfast , Northern Ireland
Bio-protocol author page: a4447
 and Anna D. Krasnodembskaya
Anna D. KrasnodembskayaAffiliation: Centre for Experimental Medicine, School of Medicine, Dentistry and Biomedical Sciences, Queen’s University Belfast, Belfast , Northern Ireland
For correspondence: a.krasnodembskaya@qub.ac.uk
Bio-protocol author page: a4449
date: 5/5/2017, 189 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2255.

Brief version appeared in Stem Cells, Aug 2016
Mesenchymal stem/stromal cells (MSC) are adult stem cells which have been shown to improve survival, enhance bacterial clearance and alleviate inflammation in pre-clinical models of acute respiratory distress syndrome (ARDS) and sepsis. These diseases are characterised by uncontrolled inflammation often underpinned by bacterial infection. The mechanisms of MSC immunomodulatory effects are not fully understood yet. We sought to investigate MSC cell contact-dependent communication with alveolar macrophages (AM), professional phagocytes which play an important role in the lung inflammatory responses and anti-bacterial defence. With the use of a basic direct co-culture system, confocal microscopy and flow cytometry we visualised and effectively quantified MSC mitochondrial transfer to AM through tunnelling nanotubes (TNT). To model the human AM, primary monocytes were isolated from human donor blood and differentiated into macrophages (monocyte derived macrophages, MDM) in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF), thus allowing adaptation of an AM-like phenotype (de Almeida et al., 2000; Guilliams et al., 2013). Human bone-marrow derived MSC, were labelled with mitochondria-specific fluorescent stain, washed extensively, seeded into the tissue culture plate with MDMs at the ratio of 1:20 (MSC/MDM) and co-cultured for 24 h. TNT formation and mitochondrial transfer were visualised by confocal microscopy and semi-quantified by flow cytometry. By using the method we described here we established that MSC use TNTs as the means to transfer mitochondria to macrophages. Further studies demonstrated that mitochondrial transfer enhances macrophage oxidative phosphorylation and phagocytosis. When TNT formation was blocked by cytochalasin B, MSC effect on macrophage phagocytosis was completely abrogated. This is the first study to demonstrate TNT-mediated mitochondrial transfer from MSC to innate immune cells.

Metabolic Heavy Isotope Labeling to Study Glycerophospholipid Homeostasis of Cultured Cells

Featured protocol,  Authors: Satu Hänninen
Satu HänninenAffiliation: Department of Biochemistry and Developmental Biology, Medical Faculty, University of Helsinki, Helsinki, Finland
For correspondence: satu.m.hanninen@helsinki.fi
Bio-protocol author page: a4478
Pentti Somerharju
Pentti SomerharjuAffiliation: Department of Biochemistry and Developmental Biology, Medical Faculty, University of Helsinki, Helsinki, Finland
Bio-protocol author page: a4479
 and Martin Hermansson
Martin HermanssonAffiliation: Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark
Bio-protocol author page: a4480
date: 5/5/2017, 163 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2268.

Brief version appeared in Biochim Biophys Acta, Sep 2016
Glycerophospholipids consist of a glycerophosphate backbone to which are esterified two acyl chains and a polar head group. The head group (e.g., choline, ethanolamine, serine or inositol) defines the glycerophospholipid class, while the acyl chains together with the head group define the glycerophospholipid molecular species. Stable heavy isotope (e.g., deuterium)-labeled head group precursors added to the culture medium incorporate efficiently into glycerophospholipids of mammalian cells, which allows one to determine the rates of synthesis, acyl chain remodeling or turnover of the individual glycerophospholipids using mass spectrometry. This protocol describes how to study the metabolism of the major mammalian glycerophospholipids i.e., phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines and phosphatidylinositols with this approach.

In vitro Assays for Measuring Endothelial Permeability by Transwells and Electrical Impedance Systems

Featured protocol,  Authors: Hong-Ru Chen
Hong-Ru ChenAffiliation: The Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan City, Taiwan
Bio-protocol author page: a4488
 and Trai-Ming Yeh
Trai-Ming YehAffiliation 1: The Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan City, Taiwan
Affiliation 2: Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, Tainan City, Taiwan
For correspondence: today@mail.ncku.edu.tw
Bio-protocol author page: a4489
date: 5/5/2017, 160 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2273.

Brief version appeared in PLoS Negl Trop Dis, Jul 2016
Vascular leakage is an important feature in several diseases, such as septic shock, viral hemorrhagic fever, cancer metastasis and ischemia-reperfusion injuries. Thus establishing assays for measuring endothelial permeability will provide insight into the establishment or progression of such diseases. Here, we provide transwell permeability assay and electrical impedance sensing assay for studying endothelial permeability in vitro. With these methods, the effect of a molecule on endothelial permeability could be defined.

Explant Methodology for Analyzing Neuroblast Migration

Featured protocol,  Authors: Kirsty J. Dixon
Kirsty J. DixonAffiliation: Department of Transplant Surgery, Virginia Commonwealth University, Richmond, USA
Bio-protocol author page: a4427
Alisa Turbic
Alisa TurbicAffiliation: Department of Epidemiology and Preventive Medicine, Monsah University, Melbourne, Australia
Bio-protocol author page: a4428
Ann M. Turnley
Ann M. TurnleyAffiliation: Department of Anatomy and Neuroscience, The University of Melbourne, Parkville, Australia
Bio-protocol author page: a4429
 and Daniel J. Liebl
Daniel J. LieblAffiliation: The Miami Project to Cure Paralysis and Department of Neurological Surgery, University of Miami, Miami, USA
For correspondence: dliebl@miami.edu
Bio-protocol author page: a4430
date: 5/5/2017, 144 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2249.

Brief version appeared in Stem Cell Res, Nov 2016
The subventricular zone (SVZ) in the mammalian forebrain contains stem/progenitor cells that migrate through the rostral migratory stream (RMS) to the olfactory bulb throughout adulthood. SVZ-derived explant cultures provide a convenient method to assess factors regulating the intermediary stage of neural stem/progenitor cell migration. Here, we describe the isolation of SVZ-derived RMS explants from the neonatal mouse brain, and the conditions required to culture and evaluate their migration.

Phos-tag Immunoblot Analysis for Detecting IRF5 Phosphorylation

Authors: Go R. Sato
Go R. SatoAffiliation: Department of Immunology, Yokohama City University Graduate School of Medicine, Yokohama, Japan
Bio-protocol author page: a4548
Tatsuma Ban
Tatsuma BanAffiliation: Department of Immunology, Yokohama City University Graduate School of Medicine, Yokohama, Japan
For correspondence: tatban@yokohama-cu.ac.jp
Bio-protocol author page: a4549
 and Tomohiko Tamura
Tomohiko TamuraAffiliation: Department of Immunology, Yokohama City University Graduate School of Medicine, Yokohama, Japan
For correspondence: tamurat@yokohama-cu.ac.jp
Bio-protocol author page: a4550
date: 5/20/2017, 126 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2295.

[Abstract] While the activation of the transcription factor interferon regulatory factor 5 (IRF5) is critical for the induction of innate immune responses, it also contributes to the pathogenesis of the autoimmune disease systemic lupus erythematosus (SLE). IRF5 phosphorylation is a hallmark of its activation ...

Assaying the Effects of Splice Site Variants by Exon Trapping in a Mammalian Cell Line

Authors: Stuart W. Tompson
Stuart W. TompsonAffiliation: Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison, Madison, Wisconsin, USA
For correspondence: stompson@wisc.edu
Bio-protocol author page: a4509
 and Terri L. Young
Terri L. YoungAffiliation: Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison, Madison, Wisconsin, USA
Bio-protocol author page: a4510
date: 5/20/2017, 98 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2281.

[Abstract] There are several in silico programs that endeavor to predict the functional impact of an individual’s sequence variation at splice donor/acceptor sites, but experimental confirmation is problematic without a source of RNA from the individual that carries the variant. With the aid of an exon trapping ...

Spore Preparation Protocol for Enrichment of Clostridia from Murine Intestine

Authors: Eric M. Velazquez
Eric M. VelazquezAffiliation: Department of Medical Microbiology and Immunology, School of Medicine, University of California at Davis, One Shields Avenue, Davis, CA 95616, USA
Bio-protocol author page: a4551
Fabian Rivera-Chávez
Fabian Rivera-ChávezAffiliation: Department of Medical Microbiology and Immunology, School of Medicine, University of California at Davis, One Shields Avenue, Davis, CA 95616, USA
Bio-protocol author page: a4552
 and Andreas J. Bäumler
Andreas J. BäumlerAffiliation: Department of Medical Microbiology and Immunology, School of Medicine, University of California at Davis, One Shields Avenue, Davis, CA 95616, USA
For correspondence: ajbaumler@ucdavis.edu
Bio-protocol author page: a4553
date: 5/20/2017, 168 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2296.

[Abstract] In recent years, many spore-forming commensal Clostridia found in the gut have been discovered to promote host physiology, immune development, and protection against infections. We provide a detailed protocol for rapid enrichment of spore-forming bacteria from murine intestine. Briefly, contents from ...

Creating a RAW264.7 CRISPR-Cas9 Genome Wide Library

Authors: Brooke A Napier
Brooke A Napier Affiliation: Department of Microbiology and Immunology, Stanford School of Medicine, Stanford University, Stanford, CA, USA
Bio-protocol author page: a4566
 and Denise M Monack
Denise M MonackAffiliation: Department of Microbiology and Immunology, Stanford School of Medicine, Stanford University, Stanford, CA, USA
For correspondence: dmonack@stanford.edu
Bio-protocol author page: a4567
date: 5/20/2017, 112 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2320.

[Abstract] The bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 genome editing tools are used in mammalian cells to knock-out specific genes of interest to elucidate gene function. The CRISPR-Cas9 system requires that the mammalian cell expresses Cas9 endonuclease, guide RNA (gRNA) ...

Detection of ASC Oligomerization by Western Blotting

Authors: Jérôme Lugrin
Jérôme LugrinAffiliation: Department of Biochemistry, University of Lausanne, CH-1066 Epalinges, Switzerland
Bio-protocol author page: a4538
 and Fabio Martinon
Fabio Martinon Affiliation: Department of Biochemistry, University of Lausanne, CH-1066 Epalinges, Switzerland
For correspondence: Fabio.Martinon@unil.ch
Bio-protocol author page: a4539
date: 5/20/2017, 95 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2292.

[Abstract] The apoptosis-associated speck-like protein with a caspase-recruitment domain (ASC) adaptor protein bridges inflammasome sensors and caspase-1. Upon inflammasome activation, ASC nucleates in a prion-like manner into a large and single platform responsible for the recruitment and the activation of caspase-1. ...

Muscle Histology Characterization Using H&E Staining and Muscle Fiber Type Classification Using Immunofluorescence Staining

Authors: Chao Wang
Chao WangAffiliation: Department of Animal Science, Purdue University, West Lafayette, Indiana, USA
For correspondence: wang1438@purdue.edu
Bio-protocol author page: a4505
Feng Yue
Feng YueAffiliation: Department of Animal Science, Purdue University, West Lafayette, Indiana, USA
Bio-protocol author page: a4506
 and Shihuan Kuang
Shihuan KuangAffiliation 1: Department of Animal Science, Purdue University, West Lafayette, Indiana, USA
Affiliation 2: Center for Cancer Research, Purdue University, West Lafayette, Indiana, USA
For correspondence: skuang@purdue.edu
Bio-protocol author page: a2572
date: 5/20/2017, 111 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2279.

[Abstract] Muscle function is determined by its structure and fiber type composition. Here we describe a protocol to examine muscle histology and myofiber types using hematoxylin and eosin (H&E) and immunofluorescence staining, respectively. H&E stain nucleus in blue and cytoplasm in red, therefore allowing for morphological analyses, such as myofiber diameter, the presence of degenerated and regenerated myofibers, and adipocytes and fibrotic cells. Muscle fibers in adult skeletal muscles of rodents are classified into 4 subtypes based on the expression of myosin heavy chain proteins: Myh7 (type I fiber), Myh2 (type IIA fiber), Myh1 (type IIX fiber), Myh4 (type IIB fiber). A panel of monoclonal antibodies can be used to specifically label these muscle fiber subtypes. These protocols are commonly used in the study of muscle development, growth and regeneration (for example: Wang et al., 2015; Nie et al., 2016; Yue et al., 2016; Wang et al., 2017)....

Isolation of Murine Alveolar Type II Epithelial Cells

Authors: Fan Sun
Fan SunAffiliation 1: University of Pittsburgh Cancer Institute, University of Pittsburgh, Pittsburgh, USA
Affiliation 2: Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, USA
Bio-protocol author page: a4527
Gutian Xiao
Gutian XiaoAffiliation 1: University of Pittsburgh Cancer Institute, University of Pittsburgh, Pittsburgh, USA
Affiliation 2: Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, USA
Bio-protocol author page: a4528
 and Zhaoxia Qu
Zhaoxia QuAffiliation 1: University of Pittsburgh Cancer Institute, University of Pittsburgh, Pittsburgh, USA
Affiliation 2: Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, USA
For correspondence: quz@upmc.edu
Bio-protocol author page: a4529
date: 5/20/2017, 125 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2288.

[Abstract] We have optimized a protocol for isolation of alveolar type II epithelial cells from mouse lung. Lung cell suspensions are prepared by intratracheal instillation of dispase and agarose followed by mechanical disaggregation of the lungs. Alveolar type II epithelial cells are purified from these lung ...

Murine Bronchoalveolar Lavage

Authors: Fan Sun
Fan SunAffiliation 1: University of Pittsburgh Cancer Institute, University of Pittsburgh, Pittsburgh, USA
Affiliation 2: Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, USA
Bio-protocol author page: a4527
Gutian Xiao
Gutian XiaoAffiliation 1: University of Pittsburgh Cancer Institute, University of Pittsburgh, Pittsburgh, USA
Affiliation 2: Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, USA
Bio-protocol author page: a4528
 and Zhaoxia Qu
Zhaoxia QuAffiliation 1: University of Pittsburgh Cancer Institute, University of Pittsburgh, Pittsburgh, USA
Affiliation 2: Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, USA
For correspondence: quz@upmc.edu
Bio-protocol author page: a4529
date: 5/20/2017, 117 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2287.

[Abstract] A basic Bronchoalveolar lavage (BAL) procedure in mouse is described here. Cells and fluids obtained from BAL can be analyzed by Hema3-staining, immunostaining, Fluorescence-activated cell sorting (FACS), PCR, bicinchoninic acid protein assay, enzyme-linked immunosorbent assay (ELISA), luminex assays, ...

Analysis of in vivo Interaction between RNA Binding Proteins and Their RNA Targets by UV Cross-linking and Immunoprecipitation (CLIP) Method

Authors: Pamela Bielli
Pamela BielliAffiliation 1: Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy
Affiliation 2: Laboratory of Neuroembriology, Fondazione Santa Lucia, Rome, Italy
Bio-protocol author page: a4492
 and Claudio Sette
Claudio SetteAffiliation 1: Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy
Affiliation 2: Laboratory of Neuroembriology, Fondazione Santa Lucia, Rome, Italy
For correspondence: claudio.sette@uniroma2.it
Bio-protocol author page: a4493
date: 5/20/2017, 152 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2274.

[Abstract] RNA metabolism is tightly controlled across different tissues and developmental stages, and its dysregulation is one of the molecular hallmarks of cancer. Through direct binding to specific sequence element(s), RNA binding proteins (RBPs) play a pivotal role in co- and post-transcriptional RNA regulatory ...

Nucleosome Positioning Assay

Authors: Zhongliang Zhao
Zhongliang ZhaoAffiliation: Division of Molecular Biology of the Cell II, German Cancer Research Center, DKFZ-ZMBH Alliance, Im Neuenheimer Feld 581, 69120 Heidelberg, Germany
Bio-protocol author page: a4525
 and Holger Bierhoff
Holger BierhoffAffiliation 1: Department of Biochemistry, Institute of Biochemistry and Biophysics, Center for Molecular Biomedicine (CMB), Friedrich Schiller University Jena, Hans-Knöll-Str. 2, Jena, Germany
Affiliation 2: Leibniz-Institute on Aging – Fritz Lipmann Institute (FLI), Beutenbergstrasse 11, Jena, Germany
For correspondence: holger.bierhoff@uni-jena.de
Bio-protocol author page: a4526
date: 5/20/2017, 102 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2285.

[Abstract] The basic unit of chromatin is the nucleosome, a histone octamer with 147 base pairs of DNA wrapped around it. Positions of nucleosomes relative to each other and to DNA elements have a strong impact on chromatin structure and gene activity and are tightly regulated at multiple levels, i.e., DNA sequence, ...
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Scratch Wound Healing Assay

Author: Yanling Chen
Yanling ChenAffiliation: Department of Immunology, The Scripps Research Institute, La Jolla, USA
For correspondence: ylchen@scripps.edu
Bio-protocol author page: a27
date: 3/5/2012, 55889 views, 6 Q&A
DOI: https://doi.org/10.21769/BioProtoc.100.

[Abstract] The scratch wound healing assay has been widely adapted and modified to study the effects of a variety of experimental conditions, for instance, gene knockdown or chemical exposure, on mammalian cell migration and proliferation. In a typical scratch wound healing assay, a “wound gap” in a cell monolayer ...

Transwell Cell Migration Assay Using Human Breast Epithelial Cancer Cell

Author: Yanling Chen
Yanling ChenAffiliation: Department of Immunology, The Scripps Research Institute, La Jolla, USA
For correspondence: ylchen@scripps.edu
Bio-protocol author page: a27
date: 2/20/2012, 43688 views, 7 Q&A
DOI: https://doi.org/10.21769/BioProtoc.99.

[Abstract] Transwell migration assays have been widely used for studying the motility of different types of cells including metastatic cancer cells. The assay is also useful in screens for compounds that act as chemoattractants or inhibitors of chemotaxis for cells. The assay employs a permeable layer of support, ...

In vitro Culture of Human PBMCs

Authors: Santosh K Panda
Santosh K PandaAffiliation: Infectious Disease Biology, Institute of Life Sciences, Bhubaneswar, India
Bio-protocol author page: a221
 and Balachandran Ravindran
Balachandran RavindranAffiliation: Infectious Disease Biology, Institute of Life Sciences, Bhubaneswar, India
For correspondence: ravindran8@gmail.com
Bio-protocol author page: a222
date: 2/5/2013, 43493 views, 4 Q&A
DOI: https://doi.org/10.21769/BioProtoc.322.

[Abstract] Peripheral blood mononuclear cells (PBMCs) consist of chiefly of lymphocytes and monocytes. Purified PBMCs are used in vitro to evaluate a variety of functions of lymphocytes viz; a) proliferation to mitogenic (PHA, Con-A) stimulation, b) monitoring of prior sensitisation in antigen recall assays by ...

Detection of Intracellular Reactive Oxygen Species (CM-H2DCFDA)

Authors: Rabii Ameziane-El-Hassani
Rabii Ameziane-El-HassaniAffiliation 1: UBRM, Centre National de l'Energie, des Sciences et des Techniques Nucléaires, Rabat, Morocco
Affiliation 2: Institut Gustave Roussy, FRE2939 Centre National de la Recherche Scientifique, Villejuif, France
Bio-protocol author page: a200
 and Corinne Dupuy
Corinne DupuyAffiliation 1: Institut Gustave Roussy, FRE2939 Centre National de la Recherche Scientifique, Villejuif, France
Affiliation 2: University Paris, Sud 11, Orsay, France
For correspondence: dupuy@igr.fr
Bio-protocol author page: a201
date: 1/5/2013, 42366 views, 2 Q&A
DOI: https://doi.org/10.21769/BioProtoc.313.

[Abstract] Reactive oxygen species (ROS) play a critical role in cellular physiopathology. ROS are implicated in cell proliferation, signaling pathways, oxidative defense mechanisms responsible for killing of bacteria, thyroid hormonosynthesis, etc. The cellular Redox homeostasis is balanced by oxidants and antioxidants ...

[Bio101] Cell Proliferation Assay by Flow Cytometry (BrdU and PI Staining)

Author: Hui Zhu
Hui ZhuAffiliation: Department of Genetics, Stanford University, Stanford, USA
For correspondence: huizhu@stanford.edu
Bio-protocol author page: a32
date: 4/5/2012, 39658 views, 2 Q&A
DOI: https://doi.org/10.21769/BioProtoc.198.

[Abstract] Cell Proliferation assays include an important set of fluorescence-based tests that can monitor cell health and cell division by evaluating DNA synthesis through thymidine incorporation. Bromodeoxyuridine (5-bromo-2'-deoxyuridine, BrdU) is a synthetic nucleoside that is an analogue of thymidine. BrdU ...

Clonogenic Assay

Author: Xiaodong Yang
Xiaodong YangAffiliation: Department of Neurology, University of California, San Francisco, USA
For correspondence: yangxiaodong1@yahoo.com
Bio-protocol author page: a43
date: 5/20/2012, 38893 views, 5 Q&A
DOI: https://doi.org/10.21769/BioProtoc.187.

[Abstract] Clonogenic assays serve as a useful tool to test whether a given cancer therapy can reduce the clonogenic survival of tumor cells. A colony is defined as a cluster of at least 50 cells that can often only be determined microscopically. A clonogenic assay is the method of choice to determine cell reproductive ...

Isolation and Culture of Mouse Bone Marrow-derived Macrophages (BMM’phi’)

Author: Ran Chen
Ran ChenAffiliation: Department of Genetics, Stanford University, Stanford, USA
For correspondence: rcchen@jfkbio.com
Bio-protocol author page: a34
date: 2/5/2012, 35601 views, 15 Q&A
DOI: https://doi.org/10.21769/BioProtoc.68.

[Abstract] Bone marrow derived macrophages are a type of white blood cell that can be isolated from mammalian bone marrow. In this protocol, a method is described in which bone marrow cells are isolated from mouse leg bones (femur and tibia), and then differentiated to bone marrow-derived macrophages in approximately ...

In vitro Human Umbilical Vein Endothelial Cells (HUVEC) Tube-formation Assay

Authors: Josephine MY Ko
Josephine MY KoAffiliation: Clinical Oncology Department, The University of Hong Kong, Hong Kong , Hong Kong SAR
Bio-protocol author page: a100
 and Maria Li Lung
Maria Li LungAffiliation: Clinical Oncology Department, The Univerisity of Hong Kong, Hong Kong, Hong Kong SAR
For correspondence: mlilung@hku.hk
Bio-protocol author page: a101
date: 9/20/2012, 33019 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.260.

[Abstract] Angiogenesis is involved not only in pathological conditions including cancer biology and non-neoplastic diseases, but also many biological processes including reproduction, development and repair. During angiogenesis, endothelial cells (ECs) undergo activation after binding of angiogenic factors to ...

Soft–Agar colony Formation Assay

Author: FengZhi Liu
FengZhi LiuAffiliation: School of Biomedical Sciences, Thomas Jefferson University, Philadelphia, USA
For correspondence: fengzhi6@yahoo.com
Bio-protocol author page: a51
date: 7/5/2012, 31670 views, 6 Q&A
DOI: https://doi.org/10.21769/BioProtoc.220.

[Abstract] Any anchorage–independent growth of tumor cells is estimated by a soft–agar colony formation assay. This protocol provides a general workflow for establishing a soft-agar colony formation assay....

[Bio101] In vitro Differentiation of Mouse Th0, Th1 and Th2 from Naïve CD4 T Cells

Author: Jia Li
Jia LiAffiliation: Department of Immunology, Medical Center, Duke University, Durham, North Carolina, USA
For correspondence: jiali.email@gmail.com
Bio-protocol author page: a16
date: 11/20/2011, 31126 views, 18 Q&A
DOI: https://doi.org/10.21769/BioProtoc.157.

[Abstract] In vitro differentiation of helper T cells of various lineages is frequently used in T helper cell study. Naïve CD4 T cells can differentiate into certain lineage of T help cells in vitro in the presence of specific stimulatory cytokines and inhibition of cytokines that are essential for the differentiation ...
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