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Simultaneous Intranasal/Intravascular Antibody Labeling of CD4+ T Cells in Mouse Lungs

Featured protocol,  Authors: Yanqun Wang*
Yanqun WangAffiliation: State Key Laboratory of Respiratory Diseases, Guangzhou Institute of Respiratory Disease, the First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China
Bio-protocol author page: a3996
Jing Sun*
Jing SunAffiliation: State Key Laboratory of Respiratory Diseases, Guangzhou Institute of Respiratory Disease, the First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China
Bio-protocol author page: a3997
Rudragouda Channappanavar
Rudragouda ChannappanavarAffiliation: Departments of Microbiology, University of Iowa, Iowa, USA
Bio-protocol author page: a3999
Jingxian Zhao
Jingxian ZhaoAffiliation: State Key Laboratory of Respiratory Diseases, Guangzhou Institute of Respiratory Disease, the First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China
Bio-protocol author page: a3998
Stanley Perlman
Stanley PerlmanAffiliation: Departments of Microbiology, University of Iowa, Iowa, USA
For correspondence: stanley-perlman@uiowa.edu
Bio-protocol author page: a1251
 and Jincun Zhao
Jincun ZhaoAffiliation: State Key Laboratory of Respiratory Diseases, Guangzhou Institute of Respiratory Disease, the First Affiliated Hospital of Guangzhou Medical University, Guangzhou
For correspondence: zhaojincun@gird.cn
Bio-protocol author page: a1250
 (*contributed equally to this work) date: 1/5/2017, 90 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2099.

Brief version appeared in Immunity, Jun 2016
CD4+ T cell responses have been shown to be protective in many respiratory virus infections. In the respiratory tract, CD4+ T cells include cells in the airway and parenchyma and cells adhering to the pulmonary vasculature. Here we discuss in detail the methods that are useful for characterizing CD4+ T cells in different anatomic locations in mouse lungs.

In vitro Treatment of Mouse and Human Cells with Endogenous Ligands for Activation of the Aryl Hydrocarbon Receptor

Featured protocol,  Authors: Taisho Yamada
Taisho YamadaAffiliation: Division of Signaling in Cancer and Immunology, Institute for Genetic Medicine, Molecular Medical Biochemistry Unit, Biological Chemistry and Engineering Course, Graduate School of Chemical Sciences and Engineering, Hokkaido University, Sapporo, Japan
Bio-protocol author page: a3976
 and Akinori Takaoka
Akinori TakaokaAffiliation: Division of Signaling in Cancer and Immunology, Institute for Genetic Medicine, Molecular Medical Biochemistry Unit, Biological Chemistry and Engineering Course, Graduate School of Chemical Sciences and Engineering, Hokkaido University, Sapporo, Japan
For correspondence: takaoka@igm.hokudai.ac.jp
Bio-protocol author page: a2879
date: 1/5/2017, 73 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2097.

Brief version appeared in Nat Immunol, Jun 2016
Activation of the aryl hydrocarbon receptor (AHR) by endogenous ligands has been implicated in a variety of physiological processes such as cell cycle regulation, cell differentiation and immune responses. It is reported that tryptophan metabolites, such as kynurenine (Kyn) and 6-formylindolo(3,2-b)carbazole (FICZ), are endogenous ligands for AHR (Stockinger et al., 2014). This protocol is designed for treatment with Kyn or FICZ in mouse embryonic fibroblasts (MEFs) or primary peripheral monocytes.

FICZ Exposure and Viral Infection in Mice

Featured protocol,  Authors: Taisho Yamada
Taisho YamadaAffiliation: Division of Signaling in Cancer and Immunology, Institute for Genetic Medicine, Molecular Medical Biochemistry Unit, Biological Chemistry and Engineering Course, Graduate School of Chemical Sciences and Engineering, Hokkaido University, Sapporo, Japan
Bio-protocol author page: a3976
 and Akinori Takaoka
Akinori TakaokaAffiliation: Division of Signaling in Cancer and Immunology, Institute for Genetic Medicine, Molecular Medical Biochemistry Unit, Biological Chemistry and Engineering Course, Graduate School of Chemical Sciences and Engineering, Hokkaido University, Sapporo, Japan
For correspondence: takaoka@igm.hokudai.ac.jp
Bio-protocol author page: a2879
date: 1/5/2017, 79 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2096.

Brief version appeared in Nat Immunol, Jun 2016
The aryl hydrocarbon receptor (AHR) is known as a sensor for dioxins that mediates their toxicity, and also has important biophysiological roles such as circadian rhythms, cell differentiation and immune responses. 6-formylindolo(3,2-b)carbazole (FICZ), which is derived through the metabolism of L-tryptophan by ultraviolet B irradiation, is one of putative physiological ligands for AHR (Smirnova et al., 2016). It has recently been shown that endogenously-activated AHR signaling modulates innate immune response during viral infection (Yamada et al., 2016). This section describes how to treat mice with FICZ and to infect them with virus.

Primary Culture of Mouse Neurons from the Spinal Cord Dorsal Horn

Featured protocol,  Authors: De-Li Cao
De-Li CaoAffiliation: Institute of Nautical Medicine, Nantong University, Jiangsu, China
Bio-protocol author page: a3992
Peng-Bo Jing
Peng-Bo JingAffiliation: Institute of Nautical Medicine, Nantong University, Jiangsu, China
Bio-protocol author page: a3742
Bao-Chun Jiang
Bao-Chun JiangAffiliation: Institute of Nautical Medicine, Nantong University, Jiangsu, China
Bio-protocol author page: a3994
 and Yong-Jing Gao
Yong-Jing GaoAffiliation 1: Institute of Nautical Medicine, Nantong University, Jiangsu, China
Affiliation 2: Co-innovation Center of Neuroregeneration, Nantong University, Jiangsu, China
For correspondence: gaoyongjing@hotmail.com
Bio-protocol author page: a3743
date: 1/5/2017, 74 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2098.

Brief version appeared in J Clin Invest, Feb 2016
Primary afferents of sensory neurons mainly terminate in the spinal cord dorsal horn, which has an important role in the integration and modulation of sensory-related signals. Primary culture of mouse spinal dorsal horn neuron (SDHN) is useful for studying signal transmission from peripheral nervous system to the brain, as well as for developing cellular disease models, such as pain and itch. Because of the specific features of SDHN, it is necessary to establish a reliable culture method that is suitable for testing neural response to various external stimuli in vitro.

In vivo Efficacy Studies in Cell Line and Patient-derived Xenograft Mouse Models

Featured protocol,  Authors: Elizabeth A. Tovar
Elizabeth A. TovarAffiliation: Center for Cancer and Cell Biology, Van Andel Research Institute, Grand Rapids, Michigan, USA
Bio-protocol author page: a3966
Curt J. Essenburg
Curt J. EssenburgAffiliation: Center for Cancer and Cell Biology, Van Andel Research Institute, Grand Rapids, Michigan, USA
Bio-protocol author page: a3967
 and Carrie Graveel
Carrie GraveelAffiliation: Center for Cancer and Cell Biology, Van Andel Research Institute, Grand Rapids, Michigan, USA
For correspondence: carrie.graveel@vai.org
Bio-protocol author page: a3968
date: 1/5/2017, 87 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2100.

Brief version appeared in Clin Cancer Res, Feb 2016
In vivo xenograft models derived from human cancer cells have been a gold standard for evaluating the genetic drivers of cancer and are valuable preclinical models for evaluating the efficacy of cancer therapeutics. Recently, patient-derived tumorgrafts from multiple tumor types have been developed and shown to more accurately recapitulate the molecular and histological heterogeneity of cancer. Here we detail the procedures for developing patient-derived xenograft models from breast cancer tissue, cell-based xenograft models, serial tumor transplantation, tumor measurement, and drug treatment.

Measuring Procaspase-8 and -10 Processing upon Apoptosis Induction

Featured protocol,  Authors: Sabine Pietkiewicz
Sabine PietkiewiczAffiliation: Translational Inflammation Research, Institute of Experimental Internal Medicine, Otto von Guericke University Magdeburg, Medical Faculty, Magdeburg, Germany
Present address: Medical advisory service of social health insurance, Essen, Germany
Bio-protocol author page: a3934
Clara Wolfe
Clara WolfeAffiliation: Translational Inflammation Research, Institute of Experimental Internal Medicine, Otto von Guericke University Magdeburg, Medical Faculty, Magdeburg, Germany
Bio-protocol author page: a3935
Jörn H. Buchbinder
Jörn H. BuchbinderAffiliation: Translational Inflammation Research, Institute of Experimental Internal Medicine, Otto von Guericke University Magdeburg, Medical Faculty, Magdeburg, Germany
Bio-protocol author page: a3936
 and Inna N. Lavrik
Inna N. LavrikAffiliation 1: Translational Inflammation Research, Institute of Experimental Internal Medicine, Otto von Guericke University Magdeburg, Medical Faculty, Magdeburg, Germany
Affiliation 2: Federal research center Institute of Cytology and Genetics, Novosibirsk, Russia
For correspondence: inna.lavrik@med.ovgu.de
Bio-protocol author page: a3937
date: 1/5/2017, 97 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2081.

Brief version appeared in Cell Death Differ, Apr 2016
Apoptosis or programmed cell death is important for multicellular organisms to keep cell homeostasis and for the clearance of mutated or infected cells. Apoptosis can be induced by intrinsic or extrinsic stimuli. The first event in extrinsic apoptosis is the formation of the Death-Inducing Signalling Complex (DISC), where the initiator caspases-8 and -10 are fully activated by several proteolytic cleavage steps and induce the caspase cascade leading to apoptotic cell death. Analysing the processing of procaspases-8 and -10 by Western blot is a commonly used method to study the induction of apoptosis by death receptor stimulation. To analyse procaspase-8 and -10 cleavage, cells are stimulated with a death ligand for different time intervals, lysed and subjected to Western blot analysis using anti-caspase-8 and anti-caspase-10 antibodies. This allows monitoring the caspase cleavage products and thereby induction of apoptosis.

Generation of Tumour-stroma Minispheroids for Drug Efficacy Testing

Featured protocol,  Authors: Mark Watters
Mark Watters Affiliation: Apoptosis Research Centre, School of Natural Sciences, National University of Ireland, Galway, Ireland
Bio-protocol author page: a3964
 and Eva Szegezdi
Eva SzegezdiAffiliation: Apoptosis Research Centre, School of Natural Sciences, National University of Ireland, Galway, Ireland
For correspondence: eva.szegezdi@nuigalway.ie
Bio-protocol author page: a3965
date: 1/5/2017, 88 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2091.

Brief version appeared in Oncogene, Mar 2016
The three-dimensional organisation of cells in a tissue and their interaction with adjacent cells and extracellular matrix is a key determinant of cellular responses, including how tumour cells respond to stress conditions or therapeutic drugs (Elliott and Yuan, 2011). In vivo, tumour cells are embedded in a stroma formed primarily by fibroblasts that produce an extracellular matrix and enwoven with blood vessels. The 3D mixed cell type spheroid model described here incorporates these key features of the tissue microenvironment that in vivo tumours exist in; namely the three-dimensional organisation, the most abundant stromal cell types (fibroblasts and endothelial cells), and extracellular matrix. This method combined with confocal microscopy can be a powerful tool to carry out drug sensitivity, angiogenesis and cell migration/invasion assays of different tumour types.

Relative Stiffness Measurements of Cell-embedded Hydrogels by Shear Rheology in vitro

Featured protocol,  Authors: 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, University of New South Wales, Sydney, Australia
For correspondence: t.cox@garvan.org.au
Bio-protocol author page: a3986
 and 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
date: 1/5/2017, 110 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2101.

Brief version appeared in EMBO Rep, Oct 2015
Hydrogel systems composed of purified extracellular matrix (ECM) components (such as collagen, fibrin, Matrigel, and methylcellulose) are a mainstay of cell and molecular biology research. They are used extensively in many applications including tissue regeneration platforms, studying organ development, and pathological disease models such as cancer. Both the biochemical and biomechanical properties influence cellular and tissue compatibility, and these properties are altered in pathological disease progression (Cox and Erler, 2011; Bonnans et al., 2014). The use of cell-embedded hydrogels in disease models such as cancer, allow the interrogation of cell-induced changes in the biomechanics of the microenvironment (Madsen et al., 2015). Here we report a simple method to measure these cell-induced changes in vitro using a controlled strain rotational rheometer.

Ex vivo Culture of Fetal Mouse Gastric Epithelial Progenitors

Featured protocol,  Authors: Valeria Fernandez Vallone
Valeria Fernandez ValloneAffiliation: Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM), Faculty of Medicine, Université Libre de Bruxelles ULB, Brussels, Belgium
Bio-protocol author page: a3957
Morgane Leprovots
Morgane LeprovotsAffiliation: Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM), Faculty of Medicine, Université Libre de Bruxelles ULB, Brussels, Belgium
Bio-protocol author page: a3956
Gilbert Vassart
Gilbert VassartAffiliation: Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM), Faculty of Medicine, Université Libre de Bruxelles ULB, Brussels, Belgium
Bio-protocol author page: a3958
 and Marie-Isabelle Garcia
Marie-Isabelle GarciaAffiliation: Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM), Faculty of Medicine, Université Libre de Bruxelles ULB, Brussels, Belgium
For correspondence: mgarcia@ulb.ac.be
Bio-protocol author page: a3959
date: 1/5/2017, 91 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2089.

Brief version appeared in Development, May 2016
Isolation and tridimensional culture of murine fetal progenitors from the digestive tract represents a new approach to study the nature and the biological characteristics of these epithelial cells that are present before the onset of the cytodifferentiation process during development. In 2013, Mustata et al. described the isolation of intestinal fetal progenitors growing as spheroids in the ex vivo culture system initially implemented by Sato et al. (2009) to grow adult intestinal stem cells. Noteworthy, fetal-derived spheroids have high self-renewal capacity making easy their indefinite maintenance in culture. Here, we report an adapted protocol for isolation and ex vivo culture and maintenance of fetal epithelial progenitors from distal pre-glandular stomach growing as gastric spheroids (Fernandez Vallone et al., 2016).

Ex vivo Culture of Adult Mouse Antral Glands

Featured protocol,  Authors: Valeria Fernandez Vallone
Valeria Fernandez ValloneAffiliation: Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM), Faculty of Medicine, Université Libre de Bruxelles ULB, Brussels, Belgium
Bio-protocol author page: a3957
Morgane Leprovots
Morgane LeprovotsAffiliation: Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM), Faculty of Medicine, Université Libre de Bruxelles ULB, Brussels, Belgium
Bio-protocol author page: a3956
Gilbert Vassart
Gilbert VassartAffiliation: Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM), Faculty of Medicine, Université Libre de Bruxelles ULB, Brussels, Belgium
Bio-protocol author page: a3958
 and Marie-Isabelle Garcia
Marie-Isabelle GarciaAffiliation: Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM), Faculty of Medicine, Université Libre de Bruxelles ULB, Brussels, Belgium
For correspondence: mgarcia@ulb.ac.be
Bio-protocol author page: a3959
date: 1/5/2017, 81 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2088.

Brief version appeared in Development, May 2016
The tri-dimensional culture, initially described by Sato et al. (2009) in order to isolate and characterize epithelial stem cells of the adult small intestine, has been subsequently adapted to many different organs. One of the first examples was the isolation and culture of antral stem cells by Barker et al. (2010), who efficiently generated organoids that recapitulate the mature pyloric epithelium in vitro. This ex vivo approach is suitable and promising to study gastric function in homeostasis as well as in disease. We have adapted Barker’s protocol to compare homeostatic and regenerating tissues and here, we meticulously describe, step by step, the isolation and culture of antral glands as well as the isolation of single cells from antral glands that might be useful for culture after cell sorting as an example (Fernandez Vallone et al., 2016).

Optogenetic Mapping of Synaptic Connections in Mouse Brain Slices to Define the Functional Connectome of Identified Neuronal Populations

Featured protocol,  Authors: Susana Mingote
Susana MingoteAffiliation 1: Department of Psychiatry, Columbia University, New York, USA
Affiliation 2: Department of Molecular Therapeutics, NYS Psychiatric Institute, New York, USA
Bio-protocol author page: a3960
Nao Chuhma
Nao ChuhmaAffiliation 1: Department of Psychiatry, Columbia University, New York, USA
Affiliation 2: Department of Molecular Therapeutics, NYS Psychiatric Institute, New York, USA
Bio-protocol author page: a3961
 and Stephen Rayport
Stephen RayportAffiliation 1: Department of Psychiatry, Columbia University, New York, USA
Affiliation 2: Department of Molecular Therapeutics, NYS Psychiatric Institute, New York, USA
For correspondence: stephen.rayport@columbia.edu
Bio-protocol author page: a3962
date: 1/5/2017, 87 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2090.

Brief version appeared in J Neurosci, Dec 2015
Functional connectivity in a neural circuit is determined by the strength, incidence, and neurotransmitter nature of its connections (Chuhma, 2015). Using optogenetics the functional synaptic connections between an identified population of neurons and defined postsynaptic target neurons may be measured systematically in order to determine the functional connectome of that identified population. Here we describe the experimental protocol used to investigate the excitatory functional connectome of ventral midbrain dopamine neurons, mediated by glutamate cotransmission (Mingote et al., 2015). Dopamine neurons are made light sensitive by injecting an adeno-associated virus (AAV) encoding channelrhodopsin (ChR2) into the ventral midbrain of DATIREScre mice. The efficacy and specificity of ChR2 expression in dopamine neurons is verified by immunofluorescence for the dopamine-synthetic enzyme tyrosine hydroxylase. Then, slice patch-clamp recordings are made from neurons in regions recipient to dopamine neuron projections and the incidence and strength of excitatory connections determined. The summary of the incidence and strength of connections in all regions recipient to dopamine neuron projections constitute the functional connectome.

Measurement of Mechanical Tension at cell-cell junctions using two-photon laser ablation

Featured protocol,  Authors: Xuan Liang
Xuan LiangAffiliation: Divisions of Cell Biology and Molecular Medicine, Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Brisbane, Australia
Bio-protocol author page: a3845
Magdalene Michael
Magdalene MichaelAffiliation: Randall Division of Cell and Molecular Biophysics, King’s College London, Guy’s Campus, London, UK
Bio-protocol author page: a3844
 and Guillermo A. Gomez
Guillermo A. GomezAffiliation: Divisions of Cell Biology and Molecular Medicine, Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Brisbane, Australia
For correspondence: g.gomez@uq.edu.au
Bio-protocol author page: a910
date: 12/20/2016, 211 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2068.

Brief version appeared in Dev Cell, Apr 2016
The cortical actomyosin cytoskeleton is found in all non-muscle cells where a key function is to control mechanical force (Salbreux et al., 2012). When coupled to E-cadherin cell-cell adhesion, cortical actomyosin generates junctional tension that influences many aspects of tissue function, organization and morphogenesis (Lecuit and Yap, 2015). Uncovering the molecular mechanisms underlying the generation of junctional tension requires tools for measuring it in live cells with a high spatio-temporal resolution. For this, we have set up a technique of laser ablation, in which we use the high power output of a two-photon laser to physically cut the actin cortex at the sites of cell-cell adhesion labeled with E-cadherin-GFP. Tension, thus is visualized as the outwards recoil of the vertices that define a junction after this was ablated/cut. Analysis of recoil versus time allows extracting parameters related to the amount of contractile force that is applied to the junction before ablation (initial recoil) and the ratio between elasticity of the junction and viscosity of the media (cytoplasm) in which the junctional cortex is immersed. Using this approach we have discovered how Src protein-tyrosine kinase (Gomez et al., 2015); actin-binding proteins such as tropomyosins (Caldwell et al., 2014) and N-WASP (Wu et al., 2014); Myosin II (Priya et al., 2015) and coronin-1B (Michael et al., 2016) contribute to the molecular apparatus responsible for generating tension at the cell-cell junctions. This protocol describes the experimental procedure for setting up laser ablation experiments and how to optimize ablation and acquisition conditions for optimal measurements of junctional tension. It also provides a full description, step by step, of the post-acquisition analysis required to evaluate changes in contractile force as well as cell elasticity and/or cytoplasm viscosity.

Efficient AAV-mediated Gene Targeting Using 2A-based Promoter-trap System

Featured protocol,  Authors: Sivasundaram Karnan
Sivasundaram KarnanAffiliation: Department of Biochemistry, Aichi Medical University School of Medicine, Nagakute, Aichi, Japan
Bio-protocol author page: a3860
Akinobu Ota
Akinobu OtaAffiliation: Department of Biochemistry, Aichi Medical University School of Medicine, Nagakute, Aichi, Japan
Bio-protocol author page: a3861
Yuko Konishi
Yuko KonishiAffiliation: Department of Biochemistry, Aichi Medical University School of Medicine, Nagakute, Aichi, Japan
Bio-protocol author page: a3862
Md Wahiduzzaman
Md WahiduzzamanAffiliation: Department of Biochemistry, Aichi Medical University School of Medicine, Nagakute, Aichi, Japan
Bio-protocol author page: a3863
Shinobu Tsuzuki
Shinobu TsuzukiAffiliation: Department of Biochemistry, Aichi Medical University School of Medicine, Nagakute, Aichi, Japan
Bio-protocol author page: a3864
Yoshitaka Hosokawa
Yoshitaka HosokawaAffiliation: Department of Biochemistry, Aichi Medical University School of Medicine, Nagakute, Aichi, Japan
Bio-protocol author page: a3865
 and Hiroyuki Konishi
Hiroyuki KonishiAffiliation: Department of Biochemistry, Aichi Medical University School of Medicine, Nagakute, Aichi, Japan
For correspondence: hkonishi@aichi-med-u.ac.jp
Bio-protocol author page: a3866
date: 12/20/2016, 160 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2058.

Brief version appeared in Nucleic Acids Res, Apr 2016
Adeno-associated virus (AAV)-based targeting vectors have 1-4-log higher gene targeting efficiencies compared with plasmid-based targeting vectors. The efficiency of AAV-mediated gene targeting is further increased by introducing a promoter-trap system into targeting vectors. In addition, we found that the use of ribosome-skipping 2A peptide rather than commonly used internal ribosome entry site (IRES) in the promoter-trap system results in significantly higher AAV-mediated gene targeting efficiencies (Karnan et al., 2016). In this protocol, we describe the procedures for AAV-mediated gene targeting exploiting 2A for promoter trapping, including the construction of a targeting vector based on the platform plasmid pAAV-2Aneo or pAAV-2Aneo v2, production of AAV particles, infection of cells with resulting AAV-based targeting vectors, and isolation and verification of gene-targeted cell clones.

Affinity Pulldown of Biotinylated RNA for Detection of Protein-RNA Complexes

Featured protocol,  Authors: Amaresh C Panda
Amaresh C PandaAffiliation: Laboratory of Genetics and Genomics, National Institute on Aging, National Institutes of Health, 251 Bayview Blvd., Baltimore, United States
For correspondence: amaresh.panda@nih.gov
Bio-protocol author page: a3875
Jennifer L. Martindale
Jennifer L. Martindale Affiliation: Laboratory of Genetics and Genomics, National Institute on Aging, National Institutes of Health, 251 Bayview Blvd., Baltimore, United States
Bio-protocol author page: a3880
 and Myriam Gorospe
Myriam GorospeAffiliation: Laboratory of Genetics and Genomics, National Institute on Aging, National Institutes of Health, 251 Bayview Blvd., Baltimore, United States
Bio-protocol author page: a3881
date: 12/20/2016, 228 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2062.

Brief version appeared in Nucleic Acids Res, Mar 2016
RNA-binding proteins (RBPs) have recently emerged as crucial players in the regulation of gene expression. The interactions of RBPs with target mRNAs control the levels of gene products by altering different regulatory steps, including pre-mRNA splicing and maturation, nuclear mRNA export, and mRNA stability and translation (Glisovic et al., 2008). There are several methodologies available today to identify RNAs bound to specific RBPs; some detect only recombinant molecules in vitro, others detect recombinant and endogenous molecules, while others detect only endogenous molecules. Examples include systematic evolution of ligands by exponential enrichment (SELEX), biotinylated RNA pulldown assay, RNA immunoprecipitation (RIP) assay, electrophoretic mobility shift assay (EMSA), RNA footprinting analysis, and various UV crosslinking and immunoprecipitation (CLIP) methods such as CLIP, PAR-CLIP, and iCLIP (Popova et al., 2015). Here, we describe a simple and informative method to study and identify the RNA region of interaction between an RBP and its target transcript (Panda et al., 2014 and 2016). Its reproducibility and ease of use make this protocol a fast and useful method to identify interactions between RBPs and specific RNAs.

In vitro Assays for the Detection of Calreticulin Exposure, ATP and HMGB1 Release upon Cell Death

Featured protocol,  Authors: Yuting Ma
Yuting MaAffiliation 1: , Suzhou Institute of Systems Medicine, Suzhou, China
Affiliation 2: , Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China
For correspondence: yuting_ma1984@163.com
Bio-protocol author page: a3918
 and Heng Yang
Heng YangAffiliation 1: , Suzhou Institute of Systems Medicine, Suzhou, China
Affiliation 2: , Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China
Bio-protocol author page: a3919
date: 12/20/2016, 218 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2076.

Brief version appeared in Cancer Res, Sep 2015
Accumulating evidence is revealing the essential role of immune system in cancer treatment. Certain chemotherapeutic drugs can potently induce the release of ‘cell death associated molecular patterns’ (CDAMPs), which accompanies cancer cell demise. CDAMPs can engage corresponding receptors on immune cells and stimulate immune responses to achieve long-term tumor control (Ma et al., 2013; Ma et al., 2014; Yang et al., 2015). Among reported CDAMPs, calreticulin (CALR), ATP and HMGB1 are well known for their immune-stimulatory effect. Here we describe the assays that we applied to measure cell death and these CDAMPs. Briefly, cell death can be analyzed by co-staining of 4’,6-diamidino-2-phenylindole (DAPI) with 3,3’-Dihexyloxacarbocyanine Iodide [DiOC6(3)] or Annexin V. CALR exposure on the cell membrane can be detected by flow cytometry. ATP and HMGB1 release can be quantified by luminescence assay and ELISA assay respectively.

Activity-based Pull-down of Proteolytic Standard and Immunoproteasome Subunits

Featured protocol,  Authors: Tobias Baumann*
Tobias BaumannAffiliation: Comprehensive Pneumology Center (CPC), University Hospital, Ludwig-Maximilians University and Helmholtz Zentrum München, Member of the German Center for Lung Research (DZL), Munich, Germany
Bio-protocol author page: a3909
Oliver Vosyka*
Oliver VosykaAffiliation: Comprehensive Pneumology Center (CPC), University Hospital, Ludwig-Maximilians University and Helmholtz Zentrum München, Member of the German Center for Lung Research (DZL), Munich, Germany
Bio-protocol author page: a3910
Bogdan I. Florea
Bogdan I. FloreaAffiliation: Department of Bio-organic Synthesis, Leiden University, Leiden, The Netherlands
Bio-protocol author page: a3911
Hermen S. Overkleeft
Hermen S. OverkleeftAffiliation: Department of Bio-organic Synthesis, Leiden University, Leiden, The Netherlands
Bio-protocol author page: a3912
Silke Meiners
Silke MeinersAffiliation: Comprehensive Pneumology Center (CPC), University Hospital, Ludwig-Maximilians University and Helmholtz Zentrum München, Member of the German Center for Lung Research (DZL), Munich, Germany
Bio-protocol author page: a3913
 and Ilona E. Kammerl
Ilona E. KammerlAffiliation: Comprehensive Pneumology Center (CPC), University Hospital, Ludwig-Maximilians University and Helmholtz Zentrum München, Member of the German Center for Lung Research (DZL), Munich, Germany
For correspondence: ilona.kammerl@helmholtz-muenchen.de
Bio-protocol author page: a3914
 (*contributed equally to this work) date: 12/20/2016, 212 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2073.

Brief version appeared in Cell Death Differ, Jun 2016
Activity-based probes (ABP) are small organic molecules that irreversibly bind to the active center of a specific enzyme family and may be coupled to a fluorophore or an affinity tag (Li et al., 2013). Here, we describe a method to pull-down active catalytic standard and immunoproteasome subunits in cell lysates using the biotinylated, proteasome-specific ABP Biotin-Epoxomicin (Bio-EP). Covalent labeling of the active catalytic subunits with Bio-EP is followed by a pull-down using streptavidin-coated beads. After elution from the beads, enriched subunits may be detected via Western blot, tandem mass spectrometry (Li et al., 2013), or alternative techniques.

Isolation of THY1+ Undifferentiated Spermatogonia from Mouse Postnatal Testes Using Magnetic-activated Cell Sorting (MACS)

Featured protocol,  Authors: Hung-Fu Liao
Hung-Fu LiaoAffiliation: Institute of Biotechnology, National Taiwan University, Taipei, Taiwan
Bio-protocol author page: a3905
Joyce Kuo
Joyce KuoAffiliation: Graudate Institute of Anatomy and Cell Biology, National Taiwan University, College of Medicine, Taipei, Taiwan
Bio-protocol author page: a3906
Hsien-Hen Lin
Hsien-Hen LinAffiliation: Institute of Biotechnology, National Taiwan University, Taipei, Taiwan
Bio-protocol author page: a3907
 and Shau-Ping Lin
Shau-Ping LinAffiliation 1: Institute of Biotechnology, National Taiwan University, Taipei, Taiwan
Affiliation 2: Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan
Affiliation 3: Center for Systems Biology, National Taiwan University, Taipei, Taiwan
Affiliation 4: Research Center for Developmental Biology and Regenerative Medicine, National Taiwan University, Taipei, Taiwan
For correspondence: shaupinglin@ntu.edu.tw
Bio-protocol author page: a3908
date: 12/20/2016, 153 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2072.

Brief version appeared in Development, Jun 2014
In mammals, homeostasis of many tissues rely on a subpopulation of cells, referred to as stem cells, to sustain an appropriate number of undifferentiated and differentiated cells. Spermatogonial stem cells (SSCs) provide the fundamental cellular source for spermatogenesis and are responsible for the lifelong maintenance of the germline pool in testes throughout the reproductive lifespan of males. To gain insight into germline stem cell biology and develop strategies for infertility treatment, several germ cell isolation methods have been reported in order to acquire good quality and quantity of undifferentiated spermatogonia. Among them, magnetic-activated cell sorting (MACS) is an efficient cell isolation method that requires less time and less initial cell numbers to obtain an enriched cell population using an antigen-antibody reaction. Thymus cell antigen 1 (THY1, CD90.2) is recognized as a surface marker of undifferentiated spermatogonia in mouse neonatal and adult testes. Here, we describe a protocol for the isolation of one-week-old THY1+ cells and four-week-old THY1+ cells from mouse testes. The isolation procedure consists of three steps: testis collection and single cell suspension, cell labeling using a biotin-conjugated anti-THY1 antibody and magnetic cell separation. Note, this isolation protocol should be completed within five hours to maximize the quality and the amount of living cells.

Quantitative 3D Time Lapse Imaging of Muscle Progenitors in Skeletal Muscle of Live Mice

Featured protocol,  Authors: Micah T. Webster
Micah T. WebsterAffiliation: Department of Embryology, Carnegie Institution for Science, Baltimore, USA
Bio-protocol author page: a3892
Tyler Harvey
Tyler HarveyAffiliation 1: Department of Embryology, Carnegie Institution for Science, Baltimore, USA
Affiliation 2: Department of Biology, Johns Hopkins University, Baltimore, USA
Bio-protocol author page: a3893
 and Chen-Ming Fan
Chen-Ming FanAffiliation: Department of Embryology, Carnegie Institution for Science, Baltimore, USA
For correspondence: fan@ciwemb.edu
Bio-protocol author page: a3894
date: 12/20/2016, 133 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2066.

Brief version appeared in Cell Stem Cell, Feb 2016
For non-optically clear mammalian tissues, it is now possible to use multi-photon microscopy to penetrate deep into the tissue and obtain detailed single cell images in a live animal, i.e., intravital imaging. This technique is in principle applicable to any fluorescently marked cell, and we have employed it to observe stem cells during the regenerative process. Stem cell-mediated skeletal muscle regeneration in the mouse model has been classically studied at specific time points by sacrificing the animal and harvesting the muscle tissue for downstream analyses. A method for direct visualization of muscle stem cells to gain real-time information over a long period in a live mammal has been lacking. Here we describe a step-by-step protocol adapted from Webster et al. (2016) to quantitatively measure the behaviors of fluorescently labeled (GFP, EYFP) muscle stem and progenitor cells during homeostasis as well as following muscle injury.

Simultaneous Intranasal/Intravascular Antibody Labeling of CD4+ T Cells in Mouse Lungs

Authors: Yanqun Wang*
Yanqun WangAffiliation: State Key Laboratory of Respiratory Diseases, Guangzhou Institute of Respiratory Disease, the First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China
Bio-protocol author page: a3996
Jing Sun*
Jing SunAffiliation: State Key Laboratory of Respiratory Diseases, Guangzhou Institute of Respiratory Disease, the First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China
Bio-protocol author page: a3997
Rudragouda Channappanavar
Rudragouda ChannappanavarAffiliation: Departments of Microbiology, University of Iowa, Iowa, USA
Bio-protocol author page: a3999
Jingxian Zhao
Jingxian ZhaoAffiliation: State Key Laboratory of Respiratory Diseases, Guangzhou Institute of Respiratory Disease, the First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China
Bio-protocol author page: a3998
Stanley Perlman
Stanley PerlmanAffiliation: Departments of Microbiology, University of Iowa, Iowa, USA
For correspondence: stanley-perlman@uiowa.edu
Bio-protocol author page: a1251
 and Jincun Zhao
Jincun ZhaoAffiliation: State Key Laboratory of Respiratory Diseases, Guangzhou Institute of Respiratory Disease, the First Affiliated Hospital of Guangzhou Medical University, Guangzhou
For correspondence: zhaojincun@gird.cn
Bio-protocol author page: a1250
 (*contributed equally to this work) date: 1/5/2017, 90 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2099.

[Abstract] CD4+ T cell responses have been shown to be protective in many respiratory virus infections. In the respiratory tract, CD4+ T cells include cells in the airway and parenchyma and cells adhering to the pulmonary vasculature. Here we discuss in detail the methods that are useful for characterizing CD4+ ...

In vitro Treatment of Mouse and Human Cells with Endogenous Ligands for Activation of the Aryl Hydrocarbon Receptor

Authors: Taisho Yamada
Taisho YamadaAffiliation: Division of Signaling in Cancer and Immunology, Institute for Genetic Medicine, Molecular Medical Biochemistry Unit, Biological Chemistry and Engineering Course, Graduate School of Chemical Sciences and Engineering, Hokkaido University, Sapporo, Japan
Bio-protocol author page: a3976
 and Akinori Takaoka
Akinori TakaokaAffiliation: Division of Signaling in Cancer and Immunology, Institute for Genetic Medicine, Molecular Medical Biochemistry Unit, Biological Chemistry and Engineering Course, Graduate School of Chemical Sciences and Engineering, Hokkaido University, Sapporo, Japan
For correspondence: takaoka@igm.hokudai.ac.jp
Bio-protocol author page: a2879
date: 1/5/2017, 73 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2097.

[Abstract] Activation of the aryl hydrocarbon receptor (AHR) by endogenous ligands has been implicated in a variety of physiological processes such as cell cycle regulation, cell differentiation and immune responses. It is reported that tryptophan metabolites, such as kynurenine (Kyn) and 6-formylindolo(3,2-b)carbazole ...

FICZ Exposure and Viral Infection in Mice

Authors: Taisho Yamada
Taisho YamadaAffiliation: Division of Signaling in Cancer and Immunology, Institute for Genetic Medicine, Molecular Medical Biochemistry Unit, Biological Chemistry and Engineering Course, Graduate School of Chemical Sciences and Engineering, Hokkaido University, Sapporo, Japan
Bio-protocol author page: a3976
 and Akinori Takaoka
Akinori TakaokaAffiliation: Division of Signaling in Cancer and Immunology, Institute for Genetic Medicine, Molecular Medical Biochemistry Unit, Biological Chemistry and Engineering Course, Graduate School of Chemical Sciences and Engineering, Hokkaido University, Sapporo, Japan
For correspondence: takaoka@igm.hokudai.ac.jp
Bio-protocol author page: a2879
date: 1/5/2017, 79 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2096.

[Abstract] The aryl hydrocarbon receptor (AHR) is known as a sensor for dioxins that mediates their toxicity, and also has important biophysiological roles such as circadian rhythms, cell differentiation and immune responses. 6-formylindolo(3,2-b)carbazole (FICZ), which is derived through the metabolism of L-tryptophan ...

Primary Culture of Mouse Neurons from the Spinal Cord Dorsal Horn

Authors: De-Li Cao
De-Li CaoAffiliation: Institute of Nautical Medicine, Nantong University, Jiangsu, China
Bio-protocol author page: a3992
Peng-Bo Jing
Peng-Bo JingAffiliation: Institute of Nautical Medicine, Nantong University, Jiangsu, China
Bio-protocol author page: a3742
Bao-Chun Jiang
Bao-Chun JiangAffiliation: Institute of Nautical Medicine, Nantong University, Jiangsu, China
Bio-protocol author page: a3994
 and Yong-Jing Gao
Yong-Jing GaoAffiliation 1: Institute of Nautical Medicine, Nantong University, Jiangsu, China
Affiliation 2: Co-innovation Center of Neuroregeneration, Nantong University, Jiangsu, China
For correspondence: gaoyongjing@hotmail.com
Bio-protocol author page: a3743
date: 1/5/2017, 74 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2098.

[Abstract] Primary afferents of sensory neurons mainly terminate in the spinal cord dorsal horn, which has an important role in the integration and modulation of sensory-related signals. Primary culture of mouse spinal dorsal horn neuron (SDHN) is useful for studying signal transmission from peripheral nervous ...

In vivo Efficacy Studies in Cell Line and Patient-derived Xenograft Mouse Models

Authors: Elizabeth A. Tovar
Elizabeth A. TovarAffiliation: Center for Cancer and Cell Biology, Van Andel Research Institute, Grand Rapids, Michigan, USA
Bio-protocol author page: a3966
Curt J. Essenburg
Curt J. EssenburgAffiliation: Center for Cancer and Cell Biology, Van Andel Research Institute, Grand Rapids, Michigan, USA
Bio-protocol author page: a3967
 and Carrie Graveel
Carrie GraveelAffiliation: Center for Cancer and Cell Biology, Van Andel Research Institute, Grand Rapids, Michigan, USA
For correspondence: carrie.graveel@vai.org
Bio-protocol author page: a3968
date: 1/5/2017, 87 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2100.

[Abstract] In vivo xenograft models derived from human cancer cells have been a gold standard for evaluating the genetic drivers of cancer and are valuable preclinical models for evaluating the efficacy of cancer therapeutics. Recently, patient-derived tumorgrafts from multiple tumor types have been developed ...

Measuring Procaspase-8 and -10 Processing upon Apoptosis Induction

Authors: Sabine Pietkiewicz
Sabine PietkiewiczAffiliation: Translational Inflammation Research, Institute of Experimental Internal Medicine, Otto von Guericke University Magdeburg, Medical Faculty, Magdeburg, Germany
Present address: Medical advisory service of social health insurance, Essen, Germany
Bio-protocol author page: a3934
Clara Wolfe
Clara WolfeAffiliation: Translational Inflammation Research, Institute of Experimental Internal Medicine, Otto von Guericke University Magdeburg, Medical Faculty, Magdeburg, Germany
Bio-protocol author page: a3935
Jörn H. Buchbinder
Jörn H. BuchbinderAffiliation: Translational Inflammation Research, Institute of Experimental Internal Medicine, Otto von Guericke University Magdeburg, Medical Faculty, Magdeburg, Germany
Bio-protocol author page: a3936
 and Inna N. Lavrik
Inna N. LavrikAffiliation 1: Translational Inflammation Research, Institute of Experimental Internal Medicine, Otto von Guericke University Magdeburg, Medical Faculty, Magdeburg, Germany
Affiliation 2: Federal research center Institute of Cytology and Genetics, Novosibirsk, Russia
For correspondence: inna.lavrik@med.ovgu.de
Bio-protocol author page: a3937
date: 1/5/2017, 97 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2081.

[Abstract] Apoptosis or programmed cell death is important for multicellular organisms to keep cell homeostasis and for the clearance of mutated or infected cells. Apoptosis can be induced by intrinsic or extrinsic stimuli. The first event in extrinsic apoptosis is the formation of the Death-Inducing Signalling ...

Generation of Tumour-stroma Minispheroids for Drug Efficacy Testing

Authors: Mark Watters
Mark Watters Affiliation: Apoptosis Research Centre, School of Natural Sciences, National University of Ireland, Galway, Ireland
Bio-protocol author page: a3964
 and Eva Szegezdi
Eva SzegezdiAffiliation: Apoptosis Research Centre, School of Natural Sciences, National University of Ireland, Galway, Ireland
For correspondence: eva.szegezdi@nuigalway.ie
Bio-protocol author page: a3965
date: 1/5/2017, 88 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2091.

[Abstract] The three-dimensional organisation of cells in a tissue and their interaction with adjacent cells and extracellular matrix is a key determinant of cellular responses, including how tumour cells respond to stress conditions or therapeutic drugs (Elliott and Yuan, 2011). In vivo, tumour cells are embedded ...

Relative Stiffness Measurements of Cell-embedded Hydrogels by Shear Rheology in vitro

Authors: 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, University of New South Wales, Sydney, Australia
For correspondence: t.cox@garvan.org.au
Bio-protocol author page: a3986
 and 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
date: 1/5/2017, 110 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2101.

[Abstract] Hydrogel systems composed of purified extracellular matrix (ECM) components (such as collagen, fibrin, Matrigel, and methylcellulose) are a mainstay of cell and molecular biology research. They are used extensively in many applications including tissue regeneration platforms, studying organ development, ...

Ex vivo Culture of Fetal Mouse Gastric Epithelial Progenitors

Authors: Valeria Fernandez Vallone
Valeria Fernandez ValloneAffiliation: Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM), Faculty of Medicine, Université Libre de Bruxelles ULB, Brussels, Belgium
Bio-protocol author page: a3957
Morgane Leprovots
Morgane LeprovotsAffiliation: Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM), Faculty of Medicine, Université Libre de Bruxelles ULB, Brussels, Belgium
Bio-protocol author page: a3956
Gilbert Vassart
Gilbert VassartAffiliation: Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM), Faculty of Medicine, Université Libre de Bruxelles ULB, Brussels, Belgium
Bio-protocol author page: a3958
 and Marie-Isabelle Garcia
Marie-Isabelle GarciaAffiliation: Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM), Faculty of Medicine, Université Libre de Bruxelles ULB, Brussels, Belgium
For correspondence: mgarcia@ulb.ac.be
Bio-protocol author page: a3959
date: 1/5/2017, 91 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2089.

[Abstract] Isolation and tridimensional culture of murine fetal progenitors from the digestive tract represents a new approach to study the nature and the biological characteristics of these epithelial cells that are present before the onset of the cytodifferentiation process during development. In 2013, Mustata ...

Ex vivo Culture of Adult Mouse Antral Glands

Authors: Valeria Fernandez Vallone
Valeria Fernandez ValloneAffiliation: Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM), Faculty of Medicine, Université Libre de Bruxelles ULB, Brussels, Belgium
Bio-protocol author page: a3957
Morgane Leprovots
Morgane LeprovotsAffiliation: Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM), Faculty of Medicine, Université Libre de Bruxelles ULB, Brussels, Belgium
Bio-protocol author page: a3956
Gilbert Vassart
Gilbert VassartAffiliation: Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM), Faculty of Medicine, Université Libre de Bruxelles ULB, Brussels, Belgium
Bio-protocol author page: a3958
 and Marie-Isabelle Garcia
Marie-Isabelle GarciaAffiliation: Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM), Faculty of Medicine, Université Libre de Bruxelles ULB, Brussels, Belgium
For correspondence: mgarcia@ulb.ac.be
Bio-protocol author page: a3959
date: 1/5/2017, 81 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2088.

[Abstract] The tri-dimensional culture, initially described by Sato et al. (2009) in order to isolate and characterize epithelial stem cells of the adult small intestine, has been subsequently adapted to many different organs. One of the first examples was the isolation and culture of antral stem cells by Barker ...
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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, 51658 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, 41555 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, ...

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, 38163 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 ...

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, 37570 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 ...

[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, 37164 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, 34791 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, 33935 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, 30713 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, 29955 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, 29201 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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