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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.

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.

Exopolysaccharide Quantification for the Plant Pathogen Ralstonia solanacearum

Featured protocol,  Authors: Rémi Peyraud
Rémi PeyraudAffiliation: LIPM, Université de Toulouse, INRA, CNRS, Castanet-Tolosan, France
Bio-protocol author page: a4530
Timothy P. Denny
Timothy P. DennyAffiliation: Department of Plant Pathology, University of Georgia, Athens, Georgia
Bio-protocol author page: a4531
 and Stéphane Genin
Stéphane GeninAffiliation: LIPM, Université de Toulouse, INRA, CNRS, Castanet-Tolosan, France
For correspondence: Stephane.Genin@inra.fr
Bio-protocol author page: a4532
date: 5/20/2017, 98 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2289.

Brief version appeared in PLoS Pathog, Oct 2016
Soluble exopolysaccharide is a major virulence factor produced by the plant pathogen Ralstonia solanacearum. Its massive production during plant infection is associated with the arrest of water flow in xylem vessels leading eventually to plant death. The composition of this heavy macromolecule includes mainly N-acetylgalactosamine. Here we describe a colorimetric method for quantitative determination of the soluble exopolysaccharide present in culture supernatant of R. solanacearum.

1-MCP (1-methylcyclopropene) Treatment Protocol for Fruit or Vegetables

Featured protocol,  Authors: Gamrasni Dan
Gamrasni DanAffiliation 1: Fruit Storage Research Laboratory, Israel Fruit Growers’ Association, Kiryat Shmona, Israel
Affiliation 2: Tel Hai Academic College, Tel Hai, Israel
Affiliation 3: Migal Institute, Kiryat Shmona, Israel
For correspondence: danny.gamrasni@gmail.com
Bio-protocol author page: a4422
Goldway Martin
Goldway MartinAffiliation 1: Tel Hai Academic College, Tel Hai, Israel
Affiliation 2: Migal Institute, Kiryat Shmona, Israel
Bio-protocol author page: a4423
Stern Yosi
Stern YosiAffiliation: RIMI, Petah Tikva, Israel
Bio-protocol author page: a4424
Breitel Dario
Breitel DarioAffiliation: Department of Plant and Environmental Sciences, Weizmann Institute of Science, Rehovot, Israel
Bio-protocol author page: a4425
 and Aharoni Asaph
Aharoni AsaphAffiliation: Department of Plant and Environmental Sciences, Weizmann Institute of Science, Rehovot, Israel
Bio-protocol author page: a4426
date: 5/20/2017, 114 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2278.

Brief version appeared in PLoS Genet, Mar 2016
1-MCP (1-methylcyclopropene) is a simple synthetic hydrocarbon molecule that interacts with the ethylene receptor and inhibits the response of fruit or plant to ethylene. 1-MCP has opened new opportunities in handling harvested crops and serves as a powerful tool to learn about plant response to ethylene (Watkins and Miller, 2006). 1-MCP is manufactured by Agrofresh and known by its commercial name SmartfreshSM.

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.

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.

Imaging the Pharynx to Measure the Uptake of Doxorubicin in Caenorhabditis elegans

Featured protocol,  Authors: Sivathevy Amirthagunabalasingam
Sivathevy AmirthagunabalasingamAffiliation: Maisonneuve-Rosemont Hospital Research Center, and the Université de Montréal, Faculty of Medicine, Department of Medicine, Montréal, Canada
Bio-protocol author page: a4534
Arturo Papaluca
Arturo PapalucaAffiliation: , Lady Davis Institute, McGill University, Montréal, Canada
Bio-protocol author page: a4535
Taramatti Harihar
Taramatti HariharAffiliation: Maisonneuve-Rosemont Hospital Research Center, and the Université de Montréal, Faculty of Medicine, Department of Medicine, Montréal, Canada
Bio-protocol author page: a4536
 and Dindial Ramotar
Dindial RamotarAffiliation: Maisonneuve-Rosemont Hospital Research Center, and the Université de Montréal, Faculty of Medicine, Department of Medicine, Montréal, Canada
For correspondence: dindial.ramotar@umontreal.ca
Bio-protocol author page: a4537
date: 5/20/2017, 98 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2291.

Brief version appeared in Sci Rep, Oct 2016
Caenorhabditis elegans offers an array of advantages to investigate the roles of uptake transporters. Herein, an epifluorescent microscopy approach was developed to monitor the uptake of the autofluorescent anticancer drug, doxorubicin, into the pharynx of C. elegans by organic cation transporters.

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).

Simple Spectroscopic Determination of Nitrate, Nitrite, and Ammonium in Arabidopsis thaliana

Featured protocol,  Authors: Takushi Hachiya
Takushi HachiyaAffiliation 1: Department of Biological Mechanisms and Functions, Graduate School of Bioagricultural Sciences,, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8601, Japan
Affiliation 2: Institute for Advanced Research, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8602, Japan
For correspondence: takushi@agr.nagoya-u.ac.jp
Bio-protocol author page: a4507
 and Yuki Okamoto
Yuki OkamotoAffiliation: Department of Biological Mechanisms and Functions, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8601, Japan
Bio-protocol author page: a4508
date: 5/20/2017, 129 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2280.

Brief version appeared in Plant Cell Physiol, Nov 2016
Plants use nitrate, nitrite, and ammonium as inorganic nitrogen (N) sources. These N compounds are included in plant tissues at various concentrations depending on the balance between their uptake and assimilation. Thus, the contents of nitrate, nitrite, and ammonium are physiological indicators of plant N economy. Here, we describe a protocol for measurement of these inorganic N species in A. thaliana shoots or roots.

Plasma Membrane Preparation from Lilium davidii and Oryza sativa Mature and Germinated Pollen

Featured protocol,  Authors: Ning Yang
Ning YangAffiliation: Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing, China
Bio-protocol author page: a4554
Bing Han
Bing HanAffiliation: Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing, China
Bio-protocol author page: a4555
Lingtong Liu
Lingtong LiuAffiliation: Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing, China
Bio-protocol author page: a4556
Hao Yang
Hao YangAffiliation: Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing, China
Bio-protocol author page: a4557
 and Tai Wang
Tai WangAffiliation: Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing, China
For correspondence: twang@ibcas.ac.cn
Bio-protocol author page: a4558
date: 5/20/2017, 91 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2297.

Brief version appeared in J Integr Plant Biol, Dec 2010
Pollen germination is an excellent process to study cell polarity establishment. During this process, the tip-growing pollen tube will start elongating. The plasma membrane as the selectively permeable barrier that separate the inner and outer cell environment plays crucial roles in this process. This protocol described an efficient aqueous polymer two-phase system followed by alkaline solution washing to prepare Lilium davidii or Oryza sativa plasma membrane with high purity.

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.

Nuclei Isolation from Nematode Ascaris

Featured protocol,  Authors: Yuanyuan Kang
Yuanyuan KangAffiliation: Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, USA
Bio-protocol author page: a4463
Jianbin Wang
Jianbin WangAffiliation: Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, USA
Bio-protocol author page: a4464
 and Richard E. Davis
Richard E. DavisAffiliation: Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, USA
For correspondence: richard.davis@ucdenver.edu
Bio-protocol author page: a4465
date: 5/5/2017, 185 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2262.

Brief version appeared in Cell Rep, Aug 2016
Preparing nuclei is necessary in a variety of experimental paradigms to study nuclear processes. In this protocol, we describe a method for rapid preparation of large number of relatively pure nuclei from Ascaris embryos or tissues that are ready to be used for further experiments such as chromatin isolation and ChIP-seq, nuclear RNA analyses, or preparation of nuclear extracts (Kang et al., 2016; Wang et al., 2016).

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.

Measuring Cyanobacterial Metabolism in Biofilms with NanoSIMS Isotope Imaging and Scanning Electron Microscopy (SEM)

Featured protocol,  Authors: Rhona K. Stuart
Rhona K. StuartAffiliation: Physical and Life Sciences Division, Lawrence Livermore National Laboratory, Livermore, USA
For correspondence: stuart25@llnl.gov
Bio-protocol author page: a4466
Xavier Mayali
Xavier MayaliAffiliation: Physical and Life Sciences Division, Lawrence Livermore National Laboratory, Livermore, USA
Bio-protocol author page: a4467
Michael P. Thelen
Michael P. ThelenAffiliation: Physical and Life Sciences Division, Lawrence Livermore National Laboratory, Livermore, USA
Bio-protocol author page: a4468
Jennifer Pett-Ridge
Jennifer Pett-RidgeAffiliation: Physical and Life Sciences Division, Lawrence Livermore National Laboratory, Livermore, USA
Bio-protocol author page: a4469
 and Peter K. Weber
Peter K. WeberAffiliation: Physical and Life Sciences Division, Lawrence Livermore National Laboratory, Livermore, USA
Bio-protocol author page: a4470
date: 5/5/2017, 168 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2263.

Brief version appeared in Mbio, Jun 2016
To advance the understanding of microbial interactions, it is becoming increasingly important to resolve the individual metabolic contributions of microorganisms in complex communities. Organisms from biofilms can be especially difficult to separate, image and analyze, and methods to address these limitations are needed. High resolution imaging secondary ion mass spectrometry (NanoSIMS) generates single cell isotopic composition measurements, and can be used to quantify incorporation and exchange of an isotopically labeled substrate among individual organisms. Here, incorporation of cyanobacterial extracellular organic matter (EOM) by members of a cyanobacterial mixed species biofilm is used as a model to illustrate this method. Incorporation of stable isotope labeled (15N and 13C) EOM by two groups, cyanobacteria and associated heterotrophic microbes, are quantified. Methods for generating, preparing, and analyzing samples for quantifying uptake of stable isotope-labeled EOM in the biofilm are described.

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.

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.

Adhesion and Invasion Assay Procedure Using Caco-2 Cells for Listeria monocytogenes

Featured protocol,  Authors: Swetha Reddy
Swetha ReddyAffiliation: College of Veterinary Science, Mississippi State University, Starkville, USA
For correspondence: mswethareddy@gmail.com
Bio-protocol author page: a4476
 and Frank Austin
Frank AustinAffiliation: College of Veterinary Science, Mississippi State University, Starkville, USA
Bio-protocol author page: a4477
date: 5/5/2017, 174 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2267.

Brief version appeared in Microb Pathog, Mar 2016
Listeria monocytogenes is an important Gram-positive foodborne pathogen that is a particular problem in ready-to-eat food. It has an ability to survive in harsh conditions like refrigeration temperatures and high salt concentrations and is known to cross intestinal, placental and blood-brain barriers. Several cancerous cell lines like cervical, liver, dendritic, intestinal and macrophages have been used to study in vitro propagation and survival of listeria in human cells. Human intestinal epithelial cells have been used to study how listeria crosses the intestinal barrier and cause infection. The protocol in this articles describes the procedures to grow Caco-2 cells, maintain cells and use them for adhesion and invasion assays. During adhesion assay the cells are incubated with listeria for 30 min but in invasion assay the cell growth is arrested at several time points after infection to monitor the growth and survival rate of listeria in cells.

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 in the Toll-like receptor (TLR) pathway, where active ...

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. Active caspase-1 will in turn promote the ...

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 cell suspensions through magnetic-based negative selection ...

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, etc., to examine the immune cells, pathogens, proteins ...

Exopolysaccharide Quantification for the Plant Pathogen Ralstonia solanacearum

Authors: Rémi Peyraud
Rémi PeyraudAffiliation: LIPM, Université de Toulouse, INRA, CNRS, Castanet-Tolosan, France
Bio-protocol author page: a4530
Timothy P. Denny
Timothy P. DennyAffiliation: Department of Plant Pathology, University of Georgia, Athens, Georgia
Bio-protocol author page: a4531
 and Stéphane Genin
Stéphane GeninAffiliation: LIPM, Université de Toulouse, INRA, CNRS, Castanet-Tolosan, France
For correspondence: Stephane.Genin@inra.fr
Bio-protocol author page: a4532
date: 5/20/2017, 98 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2289.

[Abstract] Soluble exopolysaccharide is a major virulence factor produced by the plant pathogen Ralstonia solanacearum. Its massive production during plant infection is associated with the arrest of water flow in xylem vessels leading eventually to plant death. The composition of this heavy macromolecule includes mainly N-acetylgalactosamine. Here we describe ...

1-MCP (1-methylcyclopropene) Treatment Protocol for Fruit or Vegetables

Authors: Gamrasni Dan
Gamrasni DanAffiliation 1: Fruit Storage Research Laboratory, Israel Fruit Growers’ Association, Kiryat Shmona, Israel
Affiliation 2: Tel Hai Academic College, Tel Hai, Israel
Affiliation 3: Migal Institute, Kiryat Shmona, Israel
For correspondence: danny.gamrasni@gmail.com
Bio-protocol author page: a4422
Goldway Martin
Goldway MartinAffiliation 1: Tel Hai Academic College, Tel Hai, Israel
Affiliation 2: Migal Institute, Kiryat Shmona, Israel
Bio-protocol author page: a4423
Stern Yosi
Stern YosiAffiliation: RIMI, Petah Tikva, Israel
Bio-protocol author page: a4424
Breitel Dario
Breitel DarioAffiliation: Department of Plant and Environmental Sciences, Weizmann Institute of Science, Rehovot, Israel
Bio-protocol author page: a4425
 and Aharoni Asaph
Aharoni AsaphAffiliation: Department of Plant and Environmental Sciences, Weizmann Institute of Science, Rehovot, Israel
Bio-protocol author page: a4426
date: 5/20/2017, 114 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2278.

[Abstract] 1-MCP (1-methylcyclopropene) is a simple synthetic hydrocarbon molecule that interacts with the ethylene receptor and inhibits the response of fruit or plant to ethylene. 1-MCP has opened new opportunities in handling harvested crops and serves as a powerful tool to learn about plant response to ethylene (Watkins and Miller, 2006). 1-MCP is manufactured ...

Lung Section Staining and Microscopy

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.

[Abstract] 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

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.

[Abstract] 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 ...

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

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.

[Abstract] 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 ...
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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] Cell Adhesion 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, 29123 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.98.

[Abstract] Cell adhesion, the binding of a cell to the extracellular matrix (ECM), other cells, or a specific surface, is essential for the growth and survival of the cell and also its communication with other cells. The process of cell adhesion involves a range of biological events such as three-dimensional re-organization ...
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