Welcome guest, Sign in

Home

Cell Biology

Isolation of Latex Bead Phagosomes from Dictyostelium for in vitro Functional Assays

Featured protocol,  Authors: Ashwin D’Souza
Ashwin D’SouzaAffiliation: Department of Biological Sciences, Tata Institute of Fundamental Research, Mumbai, India
Bio-protocol author page: a3850
Paulomi Sanghavi
Paulomi SanghaviAffiliation: Department of Biological Sciences, Tata Institute of Fundamental Research, Mumbai, India
Bio-protocol author page: a3851
Ashim Rai
Ashim RaiAffiliation: Department of Biological Sciences, Tata Institute of Fundamental Research, Mumbai, India
Bio-protocol author page: a3852
Divya Pathak
Divya PathakAffiliation: Department of Biological Sciences, Tata Institute of Fundamental Research, Mumbai, India
Bio-protocol author page: a3853
 and Roop Mallik
Roop MallikAffiliation: Department of Biological Sciences, Tata Institute of Fundamental Research, Mumbai, India
For correspondence: roop@tifr.res.in
Bio-protocol author page: a3854
date: 12/5/2016, 73 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2056.

Brief version appeared in Cell, Feb 2016
We describe a protocol to purify latex bead phagosomes (LBPs) from Dictyostelium cells. These can be later used for various in vitro functional assays. For instance, we use these LBPs to understand the microtubule motor-driven transport on in vitro polymerized microtubules. Phagosomes are allowed to mature for defined periods inside cells before extraction for in vitro motility. These assays allow us to probe how lipids on the phagosome membrane recruit and organize motors, and also measure the motion and force generation resulting from underlying lipid-motor interactions. This provides a unique opportunity to interrogate native-like organelles using biophysical and biochemical assays, and understand the role of motor proteins in phagosome maturation and pathogen clearance.

Lymphocyte Isolation, Th17 Cell Differentiation, Activation, and Staining

Featured protocol,  Authors: Pawan Kumar
Pawan KumarAffiliation: Richard King Mellon Foundation Institute for Pediatric Research, Children’s Hospital of Pittsburgh of UPMC, University of Pittsburgh, Pittsburgh, PA, USA
For correspondence: pawan.kumar3@chp.edu
Bio-protocol author page: a2949
 and Jay K Kolls
Jay K KollsAffiliation: Richard King Mellon Foundation Institute for Pediatric Research, Children’s Hospital of Pittsburgh of UPMC, University of Pittsburgh, Pittsburgh, PA, USA
For correspondence: Jay.kolls@chp.edu
Bio-protocol author page: a3831
date: 12/5/2016, 35 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2047.

Brief version appeared in Immunity, Mar 2016
In vitro Th17 (α, β T helper cell which produce IL-17A, IL-17F and IL-22) differentiation has been routinely used for functional T cells studies. Here we describe a method for Th17 cell differentiation.

Vascular Smooth Muscle Cell Isolation and Culture from Mouse Aorta

Featured protocol,  Authors: Callie S. Kwartler
Callie S. KwartlerAffiliation: Department of Internal Medicine, Division of Medical Genetics, University of Texas Health Science Center, Houston, Texas, USA
Bio-protocol author page: a3821
Ping Zhou
Ping ZhouAffiliation: Department of Internal Medicine, Division of Medical Genetics, University of Texas Health Science Center, Houston, Texas, USA
Bio-protocol author page: a3822
Shao-Qing Kuang
Shao-Qing KuangAffiliation: Department of Internal Medicine, Division of Medical Genetics, University of Texas Health Science Center, Houston, Texas, USA
Bio-protocol author page: a3823
Xue-Yan Duan
Xue-Yan DuanAffiliation: Department of Internal Medicine, Division of Medical Genetics, University of Texas Health Science Center, Houston, Texas, USA
Bio-protocol author page: a3824
Limin Gong
Limin GongAffiliation: Department of Internal Medicine, Division of Medical Genetics, University of Texas Health Science Center, Houston, Texas, USA
Bio-protocol author page: a3825
 and Dianna M. Milewicz
Dianna M. MilewiczAffiliation: Department of Internal Medicine, Division of Medical Genetics, University of Texas Health Science Center, Houston, Texas, USA
For correspondence: Dianna.M.Milewicz@uth.tmc.edu
Bio-protocol author page: a3827
date: 12/5/2016, 29 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2045.

Brief version appeared in J Clin Invest, Mar 2016
Vascular smooth muscle cells (SMC) in the ascending thoracic aorta arise from neural crest cells, whereas SMCs in the descending aorta are derived from the presomitic mesoderm. SMCs play important roles in cardiovascular development and aortic aneurysm formation. This protocol describes the detailed process for explanting ascending and descending SMCs from mouse aortic tissue. Conditions for maintenance and subculture of isolated SMCs and characterization of the vascular SMC phenotype are also described.

Analysis of Myosin II Minifilament Orientation at Epithelial Zonula Adherens

Featured protocol,  Authors: Magdalene Michael
Magdalene MichaelAffiliation: Randall Division of Cell and Molecular Biophysics, King’s College London, Guy’s Campus, London, UK
For correspondence: magdalene.michael@kcl.ac.uk
Bio-protocol author page: a3844
Xuan Liang
Xuan LiangAffiliation: Divisions of Cell Biology and Molecular Medicine, Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Brisbane, Queensland, Australia
Bio-protocol author page: a3845
 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, Queensland, Australia
For correspondence: g.gomez@uq.edu.au
Bio-protocol author page: a910
date: 12/5/2016, 37 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2054.

Brief version appeared in Dev Cell, Apr 2016
Non-muscle myosin II (NMII) form bipolar filaments, which bind F-actin to exert cellular contractility during physiological processes (Vicente-Manzanares et al., 2009). Using a combinatorial approach to fluorescently label both N- and C-termini of the NMII heavy chain, recent works have demonstrated the ability to visualize NMII bipolar filaments at various subcellular localizations (Ebrahim et al., 2013; Beach et al., 2014). At the zonula adherens (ZA) of epithelia, NMII minifilaments bind the circumferential actin bundles in a pseudo-sarcomeric manner (Ebrahim et al., 2013), a conformation required to maintain junctional tension and tissue integrity (Ratheesh et al., 2012). By expressing green fluorescent protein (GFP)-NMIIA heavy chain and immunolabel it using a NMIIA C-terminus specific antibody, we were able to visualize the NMII minifilaments bound to F-actin bundles in Caco-2 cells (Michael et al., 2016), as previously reported (Ebrahim et al., 2013; Beach et al., 2014). In addition, we designed an FIJI/MATLAB analysis module to quantify the size, distance and alignment of these minifilaments with respect to junctional F-actin at the ZA. Measurements of the dispersion of minifilaments angles were proven to be a useful parameter that closely correlated to the extent of contractility at junctions (Michael et al., 2016).

Lentiviral shRNA Screen to Identify Epithelial Integrity Regulating Genes in MCF10A 3D Culture

Featured protocol,  Authors: Elsa Marques
Elsa Marques Affiliation: Cancer Cell Circuitry Laboratory, Research Programs Unit/Translational Cancer Biology, University of Helsinki, Helsinki, Finland
Bio-protocol author page: a3834
 and Juha Klefström
Juha KlefströmAffiliation: Cancer Cell Circuitry Laboratory, Research Programs Unit/Translational Cancer Biology, University of Helsinki, Helsinki, Finland
For correspondence: juha.klefstrom@helsinki.fi
Bio-protocol author page: a3835
date: 12/5/2016, 71 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2050.

Brief version appeared in Oncogene, Mar 2016
MCF10A 3D culture system provides a reductionist model of glandular mammary epithelium which is widely used to study development of glandular architecture, the role of cell polarity and epithelial integrity in control of epithelial cell functions, and mechanisms of breast cancer. Here we describe how to use shRNA screening approach to identify critical cell pathways that couple epithelial structure to individual cell based responses such as cell cycle exit and apoptosis. These studies will help to interrogate genetic changes critical for early breast tumorigenesis. The protocol describes a library of lentiviral shRNA constructs designed to target epithelial integrity and a highly efficient method for lentiviral transduction of suspension MCF10A cultures. Furthermore, protocols are provided for setting up MCF10A 3D cultures in Matrigel for morphometric and cellular response studies via structured illumination and confocal microscopy analysis of immunostained 3D structures.

DNA Damage Induction by Laser Microirradiation

Featured protocol,  Authors: Marianna Tampere
Marianna TampereAffiliation: Science for Life Laboratory, Division of Translational Medicine and Chemical Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden
Bio-protocol author page: a3807
 and Oliver Mortusewicz
Oliver MortusewiczAffiliation: Science for Life Laboratory, Division of Translational Medicine and Chemical Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden
For correspondence: oliver.mortusewicz@scilifelab.se
Bio-protocol author page: a3808
date: 12/5/2016, 46 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2039.

Brief version appeared in Oncogene, Feb 2016
Genome instability can lead to cell death, senescence and cancerous transformation. Specific repair pathways have evolved to prevent accumulation of DNA lesions. Studying these highly dynamic and specific repair pathways requires precise spatial and temporal resolution, which can be achieved through a combination of laser microirradiaiton and live cell microscopy. DNA lesions are introduced at pre-determined sub-nuclear sites and repair can be analyzed in real time in living cells when using fluorescently tagged repair proteins (Mortusewicz et al., 2008). Alternatively, laser microirradiation can be combined with immunofluorescence analysis to study recruitment of endogenous proteins to laser-induced DNA damage tracks that can be visualized by positive controls like, e.g., γH2AX that mark sites of DNA breaks.

In vitro Chondrogenic Hypertrophy Induction of Mesenchymal Stem Cells

Featured protocol,  Authors: Sang Young Jeong
Sang Young JeongAffiliation: Biomedical Research Institute, R&D Center, MEDIPOST Co., Ltd., Gyeonggi-do, Republic of Korea
Bio-protocol author page: a3855
Miyoung Lee
Miyoung LeeAffiliation: Biomedical Research Institute, R&D Center, MEDIPOST Co., Ltd., Gyeonggi-do, Republic of Korea
Bio-protocol author page: a3856
Soo Jin Choi
Soo Jin ChoiAffiliation: Biomedical Research Institute, R&D Center, MEDIPOST Co., Ltd., Gyeonggi-do, Republic of Korea
Bio-protocol author page: a3857
Wonil Oh
Wonil OhAffiliation: Biomedical Research Institute, R&D Center, MEDIPOST Co., Ltd., Gyeonggi-do, Republic of Korea
Bio-protocol author page: a3858
 and Hong Bae Jeon
Hong Bae JeonAffiliation: Biomedical Research Institute, R&D Center, MEDIPOST Co., Ltd., Gyeonggi-do, Republic of Korea
For correspondence: jhb@medi-post.co.kr
Bio-protocol author page: a3859
date: 12/5/2016, 42 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2057.

Brief version appeared in Stem Cells, Nov 2015
To investigate underlying mechanism of chondrogenic hypertrophy, we need proper in vitro hypertrophic model of mesenchymal stem cells (MSCs). This protocol describes our defined method for induction of in vitro chondrogenic hypertrophy of human umbilical cord blood-derived MSCs (hUCB-MSCs). By adding thyroid hormone (T3; triiodothyronine) and minimum osteogenic-inducing factors to culture medium, we could induce hypertrophy of hUCB-MSCs in vitro. Hypertrophic induction was validated using immunohistochemical analysis, Western blotting and reverse transcriptase polymerase chain reaction.

Mouse Model of Dengue Virus Infection with Serotypes 1 and 2 Clinical Isolates

Featured protocol,  Authors: Satoru Watanabe
Satoru Watanabe Affiliation: Emerging Infectious Diseases, Duke-NUS Medical School, Singapore, Singapore
For correspondence: satoru.watanabe@duke-nus.edu.sg
Bio-protocol author page: a3270
Kitti Wing Ki Chan
Kitti Wing Ki ChanAffiliation: Emerging Infectious Diseases, Duke-NUS Medical School, Singapore, Singapore
Bio-protocol author page: a3810
 and Subhash G. Vasudevan
Subhash G. VasudevanAffiliation: Emerging Infectious Diseases, Duke-NUS Medical School, Singapore, Singapore
For correspondence: subhash.vasudevan@duke-nus.edu.sg
Bio-protocol author page: a3811
date: 12/5/2016, 33 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2040.

Brief version appeared in Antiviral Res, Mar 2016
Dengue is a global public health threat caused by infection with any of the 4 related dengue virus serotypes (DENV1-4). Clinical manifestations range from self-limiting febrile illness, known as dengue fever (DF), to life-threatening severe diseases, such as dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS). Most cases of DHF/DSS are associated with secondary heterotypic infections through a phenomenon that is described as antibody-dependent enhancement of infection (ADE). There are an estimated 400 million human infections and several hundred thousand cases of severe dengue occurring yearly. At present, however, there are no approved antiviral drugs against DENV infection. The lack of a suitable animal model has hampered the evaluation of novel antiviral candidates for DENV infection. Since DENV poorly establishes infection in immunocompetent mice, AG129 mice (lacking type I and II IFN [interferon] receptors) and mouse-adapted DENV2 strains have been applied to dengue animal models that enable to reproduce several of the major pathologies of human infection. Recently, we developed new mouse models with clinical isolates DENV1 and DENV2 that would be useful for drug testing and dengue pathogenesis studies (Watanabe et al., 2016). Here we describe the details to establish dengue mouse models of clinical isolates; from in vitro preparation of the materials to in vivo virus infection. Of note, since infectivity of DENV in mice differs among virus strains, not all clinical isolates can induce severe dengue.

Measurement of Intracellular Calcium Concentration in Pseudomonas aeruginosa

Featured protocol,  Authors: Manita Guragain
Manita GuragainAffiliation: Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, US
Bio-protocol author page: a3812
Anthony K. Campbell
Anthony K. CampbellAffiliation: Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, US
Bio-protocol author page: a3813
 and Marianna A. Patrauchan
Marianna A. PatrauchanAffiliation: Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, US
For correspondence: m.patrauchan@okstate.edu
Bio-protocol author page: a3814
date: 12/5/2016, 31 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2041.

Brief version appeared in J Bacteriol, Jan 2016
Characterization of the molecular mechanisms of calcium (Ca2+) regulation of bacterial physiology and virulence requires tools enabling measuring and monitoring the intracellular levels of free calcium (Ca2+in). Here, we describe a protocol optimized to use a recombinantly expressed Ca2+-binding protein, aequorin, for detecting Ca2+in in Pseudomonas aeruginosa. Upon binding to free Ca2+, aequorin undergoes chromophore oxidation and emits light, the log of which intensity linearly correlates with the amount of bound Ca2+, and therefore, can be used to measure the concentration of free Ca2+ available for binding. This protocol involves the introduction of the aequorin gene into P. aeruginosa, induction of apoaequorin production, reconstitution of the holoenzyme with its chromophore, and monitoring its luminescence. This protocol allows continuous measuring of Ca2+in concentration in vivo in response to various stimuli.

Various Modes of Spinal Cord Injury to Study Regeneration in Adult Zebrafish

Featured protocol,  Authors: Subhra Prakash Hui
Subhra Prakash HuiAffiliation 1: Department of Biophysics, Molecular Biology and Bioinformatics, University of Calcutta, Kolkata, India
Affiliation 2: Victor Chang Cardiac Research Institute, Lowy Packer Building, Darlinghurst, Australia
Bio-protocol author page: a3817
 and Sukla Ghosh
Sukla GhoshAffiliation: Department of Biophysics, Molecular Biology and Bioinformatics, University of Calcutta, Kolkata, India
For correspondence: suklagh2010@gmail.com
Bio-protocol author page: a3818
date: 12/5/2016, 42 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2043.

Brief version appeared in PLOS ONE, Dec 2015
Spinal cord injury (SCI) in mammals leads to failure of both sensory and motor functions, due to lack of axonal regrowth below the level of injury as well as inability to replace lost neural cells and to stimulate neurogenesis. In contrast, fish and amphibians are capable of regenerating a variety of their organs like limb/fin, jaw, heart and various parts of the central nervous system (CNS). Zebrafish embryo and adult has become a very popular model to study developmental biology, cell biology and regeneration for various reasons. Adult zebrafish, one of the most important vertebrate models to study regeneration, can regenerate many of their body parts like fin, jaw, heart and CNS. In the present article we provide information on how to inflict different injury modalities in adult fish spinal cord. Presently, the significant focus of mammalian SCI is to use crush and contusion injury. To generate an entity comparable to the mammalian mode of injury, we have introduced the crush model in adult zebrafish along with complete transection injury, which is also known to be a valuable model to study axonal regeneration. Here we provide full description of the highly reproducible surgical procedures including some representative results. This protocol has been adapted from our previous publications, viz. Hui et al., 2010 and Hui et al., 2014. Briefly, we have described the two different injury modalities, crush and complete transection, and demonstrated the outcome of inflicting these injuries in the adult zebrafish cord by histological analysis of the tissues.

Isolating and Measuring the Growth and Morphology of Pro-Embryogenic Masses in Araucaria angustifolia (Bertol.) Kuntze (Araucariaceae)

Featured protocol,  Authors: Jackellinne Caetano Douétts-Peres
Jackellinne Caetano Douétts-PeresAffiliation: Laboratório de Biologia Celular e Tecidual, Centro de Biociências e Biotecnologia (CBB), Universidade Estadual do Norte Fluminense Darcy Ribeiro (UENF), Campos dos Goytacazes, Rio de Janeiro, Brazil
Bio-protocol author page: a3776
Vanildo Silveira
Vanildo SilveiraAffiliation 1: Laboratório de Biotecnologia, CBB, UENF, Campos dos Goytacazes, Rio de Janeiro, Brazil
Affiliation 2: Unidade de Biologia Integrativa, Setor de Genômica e Proteômica, UENF, Campos dos Goytacazes, Rio de Janeiro, Brazil
Bio-protocol author page: a3777
Marco Antonio Lopes Cruz
Marco Antonio Lopes CruzAffiliation: Laboratório de Biotecnologia Vegetal, Núcleo em Ecologia e Desenvolvimento Sócio-ambiental de Macaé, Universidade Federal do Rio de Janeiro, Macaé, Rio de Janeiro, Brazil
Bio-protocol author page: a3778
 and Claudete Santa-Catarina
Claudete Santa-CatarinaAffiliation: Laboratório de Biologia Celular e Tecidual, Centro de Biociências e Biotecnologia (CBB), Universidade Estadual do Norte Fluminense Darcy Ribeiro (UENF), Campos dos Goytacazes, Rio de Janeiro, Brazil
For correspondence: claudete@uenf.br
Bio-protocol author page: a3779
date: 12/5/2016, 43 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2031.

Brief version appeared in PLoS One, Apr 2016
Embryogenic suspension cultures of Araucaria angustifolia (Bertol.) Kuntze (Araucariaceae) can be used as a model to test the effects of compounds added to the culture medium on the cellular growth and morphology of Pro-Embryogenic Masses (PEMs). PEMs are formed by embryogenic and suspensor-type cells. To measure changes in the cellular growth of embryogenic cultures, we performed sedimented cell volume (SCV) quantification, which is a non-destructive method. Morphological analysis by microscopy allowed for the observation of growth and development of PEMs and the alterations in embryogenic and suspensor-type cells. The methods used here provide an efficient means for monitoring the cellular growth of PEMs and identifying morphological changes during the development of embryogenic cultures. These studies can also be combined with biochemical and molecular analyses, such as proteomics, to further investigate embryo growth and morphology.

Quantitative Measurements of HIV-1 and Dextran Capture by Human Monocyte-derived Dendritic Cells (MDDCs)

Featured protocol,  Authors: Mickaël M. Ménager
Mickaël M. MénagerAffiliation: The Kimmel Center for Biology and Medicine of the Skirball Institute, New York University School of Medicine, New York, USA
For correspondence: mickael.menager@med.nyu.edu
Bio-protocol author page: a3703
 and Dan R. Littman
Dan R. LittmanAffiliation 1: The Kimmel Center for Biology and Medicine of the Skirball Institute, New York University School of Medicine, New York, USA
Affiliation 2: Howard Hughes Medical Institute, Washington, USA
Bio-protocol author page: a3704
date: 11/20/2016, 154 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2004.

Brief version appeared in Cell, Feb 2016
The aim of this protocol is to describe how to measure and quantify the amount of HIV-1 particles and dextran molecules internalized in human monocyte derived dendritic cells (MDDCs), using three different techniques: flow cytometry, quantitative PCR and confocal microscopy.

Evaluation of Cross-presentation in Bone Marrow-derived Dendritic Cells in vitro and Splenic Dendritic Cells ex vivo Using Antigen-coated Beads

Featured protocol,  Authors: Andrés Alloatti
Andrés AlloattiAffiliation: Institute Curie, PSL Research University, INSERM U932, Paris, France
For correspondence: andres.alloatti@curie.fr
Bio-protocol author page: a3727
Fiorella Kotsias
Fiorella KotsiasAffiliation 1: Institute Curie, PSL Research University, INSERM U932, Paris, France
Affiliation 2: Department of Virology, University of Buenos Aires, Buenos Aires, Argentina
Bio-protocol author page: a3728
Eik Hoffmann
Eik HoffmannAffiliation 1: Institute Curie, PSL Research University, INSERM U932, Paris, France
Affiliation 2: VIB Inflammation Research Center, Unit of Molecular Signal Transduction in Inflammation, Ghent, Belgium
Affiliation 3: Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium
Bio-protocol author page: a3725
 and Sebastian Amigorena
Sebastian AmigorenaAffiliation: Institute Curie, PSL Research University, INSERM U932, Paris, France
Bio-protocol author page: a3730
date: 11/20/2016, 140 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2015.

Brief version appeared in Immunity, Dec 2015
Antigen presentation by MHC class I molecules, also referred to as cross-presentation, elicits cytotoxic immune responses. In particular, dendritic cells (DC) are the most proficient cross-presenting cells, since they have developed unique means to control phagocytic and degradative pathways.

Analysis of Phagosomal Antigen Degradation by Flow Organellocytometry

Featured protocol,  Authors: Eik Hoffmann
Eik HoffmannAffiliation 1: Institute Curie, INSERM U932, PSL Research University, Paris, France
Affiliation 2: VIB Inflammation Research Center, Unit of Molecular Signal Transduction in Inflammation, Ghent, Belgium
Affiliation 3: Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium
For correspondence: eik.hoffmann@irc.vib-ugent.be
Bio-protocol author page: a3725
Anne-Marie Pauwels
Anne-Marie PauwelsAffiliation 1: VIB Inflammation Research Center, Unit of Molecular Signal Transduction in Inflammation, Ghent, Belgium
Affiliation 2: Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium
Bio-protocol author page: a3726
Andrés Alloatti
Andrés AlloattiAffiliation: Institute Curie, INSERM U932, PSL Research University, Paris, France
Bio-protocol author page: a3727
Fiorella Kotsias
Fiorella KotsiasAffiliation 1: Institute Curie, INSERM U932, PSL Research University, Paris, France
Affiliation 2: Department of Virology, University of Buenos Aires, Buenos Aires, Argentina
Bio-protocol author page: a3728
 and Sebastian Amigorena
Sebastian AmigorenaAffiliation: Institute Curie, INSERM U932, PSL Research University, Paris, France
Bio-protocol author page: a3730
date: 11/20/2016, 111 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2014.

Brief version appeared in Immunity, Dec 2015
Professional phagocytes internalize self and non-self particles by phagocytosis to initiate innate immune responses. After internalization, the formed phagosome matures through fusion and fission events with endosomes and lysosomes to obtain a more acidic, oxidative and hydrolytic environment for the degradation of its cargo. Interestingly, phagosome maturation kinetics differ between cell types and cell activation states. This protocol allows to quantify phagosome maturation kinetics on a single organelle level in different types of phagocytes using flow cytometry. Here, ovalbumin (OVA)-coupled particles are used as phagocytosis model system in dendritic cells (DC), which are internalized by phagocytosis. After different time points, phagosome maturation parameters, such as phagosomal degradation of OVA and acquisition of lysosomal proteins (like LAMP-1), can be measured simultaneously in a highly quantitative manner by flow organellocytometry. These read-outs can be correlated to other phagosomal functions, for example antigen degradation, processing and loading in DC.

Determination of H2O2 Generation by pHPA Assay

Featured protocol,  Authors: Jennifer L. Larson-Casey
Jennifer L. Larson-CaseyAffiliation: Department of Medicine, Division of Pulmonary, Allergy, and Critical Care Medicine, University of Alabama at Birmingham, Birmingham, AL, USA
Bio-protocol author page: a3716
 and A. Brent Carter
A. Brent CarterAffiliation 1: Department of Medicine, Division of Pulmonary, Allergy, and Critical Care Medicine, University of Alabama at Birmingham, Birmingham, AL, USA
Affiliation 2: Birmingham Veterans Administration Medical Center, Birmingham, AL, USA
For correspondence: bcarter1@uab.edu
Bio-protocol author page: a3717
date: 11/20/2016, 124 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2010.

Brief version appeared in Immunity, Mar 2016
The production of reactive oxygen species, including H2O2, is a process that can be used in signaling, cell death, or immune response. To quantify oxidative stress in cells, a fluorescence technique has been modified from a previously described method to measure H2O2 release from cells (Panus et al., 1993; Murthy et al., 2010; Larson-Casey et al., 2016; Larson-Casey et al., 2014; He et al., 2011). This assay takes advantage of H2O2-mediated oxidation of horseradish peroxidase (HRP) to Complex I, which, in turn, oxidizes p-hydroxyphenylacetic acid (pHPA) to a stable, fluorescent pHPA dimer (2,2'-dihydroxy-biphenyl-5,5’ diacetate [(pHPA)2]). The H2O2-dependent HRP-mediated oxidation of pHPA is a sensitive and specific assay for quantifying H2O2 release from cells. This assay can measure H2O2 release from whole cells, mitochondria, or the NADPH oxidase.

Microinjection of Virus into Lumbar Enlargement of Spinal Dorsal Horn in Mice

Featured protocol,  Authors: Zhi-Jun Zhang*
Zhi-Jun ZhangAffiliation 1: Institute of Nautical Medicine, Nantong University, Jiangsu, China
Affiliation 2: Department of Human Anatomy, Nantong University, Jiangsu, China
Bio-protocol author page: a3741
Peng-Bo Jing*
Peng-Bo JingAffiliation: Institute of Nautical Medicine, Nantong University, Jiangsu, China
Bio-protocol author page: a3742
 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
 (*contributed equally to this work) date: 11/20/2016, 133 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2020.

Brief version appeared in J Clin Invest, Feb 2016
In order to explore the role of a specific gene/protein in the specific segment of spinal cord, the technique of intraspinal injection is particularly used to deliver viral vectors targeting the specific gene/protein. These viral vectors can knockdown or overexpress the specific gene/protein in specific cells (glial cells or neurons). In this protocol, lentivirus containing shRNA for CXCL13 were injected into dorsal horn of the spinal lumbar enlargement segment (Jiang et al., 2016). This technique allows the study of the role of CXCL13 in the ipsilateral dorsal horn in neuropathic pain without affecting DRG or contralateral dorsal horn.

Visualising Differential Growth of Arabidopsis Epidermal Pavement Cells Using Thin Plate Spline Analysis

Featured protocol,  Authors: William Jonathan Armour
William Jonathan ArmourAffiliation: School of Life and Environmental Sciences, University of Sydney, Sydney, Australia
For correspondence: armour.william@gmail.com
Bio-protocol author page: a3748
Deborah Anne Barton
Deborah Anne BartonAffiliation: School of Life and Environmental Sciences, University of Sydney, Sydney, Australia
Bio-protocol author page: a3749
 and Robyn Lynette Overall
Robyn Lynette OverallAffiliation: School of Life and Environmental Sciences, University of Sydney, Sydney, Australia
Bio-protocol author page: a3750
date: 11/20/2016, 181 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2022.

Brief version appeared in Plant Cell, Sep 2015
Epidermal pavement cells in Arabidopsis leaves and cotyledons develop from relatively simple shapes to form complex cells that have multiple undulations of varying sizes. Analyzing the growth of individual parts of the cell wall boundaries over time is essential to understanding how pavement cells develop their complex shapes. Thin plate spline analysis is a method for visualizing the change of size and shape of objects through warping or deformation of a regular mesh and can be applied to understand cell wall growth. This protocol describes the application of thin plate spline analysis to visualize the development of individual pavement cells over time.

Isolation and Culture of Human Adipose-derived Stem Cells from Subcutaneous and Visceral White Adipose Tissue Compartments

Featured protocol,  Authors: Xiaojia Ge
Xiaojia GeAffiliation: Cell & Molecular Biology Group, Singapore Institute for Clinical Sciences (SICS), Agency for Science Technology and Research (A*STAR), Singapore, Singapore
Bio-protocol author page: a3759
Shi Chi Leow
Shi Chi LeowAffiliation: Cell & Molecular Biology Group, Singapore Institute for Clinical Sciences (SICS), Agency for Science Technology and Research (A*STAR), Singapore, Singapore
Bio-protocol author page: a3760
Durgalakshmi Sathiakumar
Durgalakshmi SathiakumarAffiliation: Cell & Molecular Biology Group, Singapore Institute for Clinical Sciences (SICS), Agency for Science Technology and Research (A*STAR), Singapore, Singapore
Bio-protocol author page: a3761
Walter Stünkel
Walter StünkelAffiliation: Cell & Molecular Biology Group, Singapore Institute for Clinical Sciences (SICS), Agency for Science Technology and Research (A*STAR), Singapore, Singapore
Bio-protocol author page: a3762
Asim Shabbir
Asim ShabbirAffiliation: Department of Surgery, National University Hospital, National University of Singapore, Singapore, Singapore
Bio-protocol author page: a3763
Jimmy Bok Yan So
Jimmy Bok Yan SoAffiliation: Department of Surgery, National University Hospital, National University of Singapore, Singapore, Singapore
Bio-protocol author page: a3764
Davide Lomanto
Davide LomantoAffiliation: Department of Surgery, National University Hospital, National University of Singapore, Singapore, Singapore
Bio-protocol author page: a3765
 and Craig McFarlane
Craig McFarlaneAffiliation: Cell & Molecular Biology Group, Singapore Institute for Clinical Sciences (SICS), Agency for Science Technology and Research (A*STAR), Singapore, Singapore
For correspondence: craig_mcfarlane@sics.a-star.edu.sg
Bio-protocol author page: a3766
date: 11/20/2016, 116 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2027.

Brief version appeared in Eukaryot Cell, Oct 2007
Human Adipose-derived Stem/Stromal Cells (ASCs) have been widely used in stem cell and obesity research, as well as clinical applications including cell-based therapies, tissue engineering and reconstruction. Compared with mesenchymal stem cells (MSCs) derived from other tissues such as umbilical cord and bone marrow, isolation of ASCs from human white adipose tissue (WAT) has great advantages due to its rich tissue source and simple surgical procedure. In this detailed protocol we describe a protocol to isolate and characterize ASCs from human WAT. Molecular characterization of isolated ASCs was performed through surface marker expression profiling using flow cytometry. Adipogenic capacity of the isolated ASCs was confirmed through inducing adipogenic differentiation and Oil Red O staining of lipid. This protocol provides researchers with the tools to culture and assess purity and adipogenic differentiation capacity of human ASCs, which can then be utilized for required downstream in vitro applications.

An in vitro Model of Neuron-macrophage Interaction to Generate Macrophages with Neurite Outgrowth Properties

Featured protocol,  Authors: Hyeok Jun Yun
Hyeok Jun YunAffiliation 1: Department of Brain Science, Ajou University School of Medicine, Suwon, Korea
Affiliation 2: BK21 PLUS Program, Department of Biomedical Sciences, Neuroscience Graduate Program, Ajou University School of Medicine, Suwon, Korea
Bio-protocol author page: a3721
 and Byung S. Kim
Byung S. KimAffiliation 1: Department of Brain Science, Ajou University School of Medicine, Suwon, Korea
Affiliation 2: BK21 PLUS Program, Department of Biomedical Sciences, Neuroscience Graduate Program, Ajou University School of Medicine, Suwon, Korea
Affiliation 3: Department of Neurology, Ajou University School of Medicine, Suwon, Korea
For correspondence: kimbg@ajou.ac.kr
Bio-protocol author page: a1956
date: 11/20/2016, 101 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2012.

Brief version appeared in J Neurosci, Dec 2015
Macrophages are known to play beneficial roles in axon regeneration after nerve injury. To develop an in vitro model in which injury signals can elicit pro-regenerative macrophage activation, we established co-cultures consisting of adult dorsal root ganglia sensory neurons and peritoneal macrophages and added cAMP analogue dibutyryl cAMP. The conditioned medium collected from the co-cultures exhibited robust neurite outgrowth activities. The neurite outgrowth activities were almost completely abrogated by addition of minocycline, a macrophage deactivator, indicating that factors responsible for neurite outgrowth are produced by activated macrophages.

Isolation and Primary Culture of Various Cell Types from Whole Human Endometrial Biopsies

Featured protocol,  Authors: Flavio Santos Vasconcelos Barros
Flavio Santos Vasconcelos BarrosAffiliation: Department of Biomedical Sciences, University of Warwick, Coventry, UK
Bio-protocol author page: a3767
Jan Joris Brosens
Jan Joris BrosensAffiliation: Department of Biomedical Sciences, University of Warwick, Coventry, UK
For correspondence: J.J.Brosens@warwick.ac.uk
Bio-protocol author page: a3768
 and Paul John Brighton
Paul John BrightonAffiliation: Department of Biomedical Sciences, University of Warwick, Coventry, UK
Bio-protocol author page: a3769
date: 11/20/2016, 102 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2028.

Brief version appeared in Stem Cells, Jul 2015
The isolation and primary culture of cells from human endometrial biopsies provides valuable experimental material for reproductive and gynaecological research. Whole endometrial biopsies are collected from consenting women and digested with collagenase and DNase I to dissociate cells from the extracellular matrix. Cell populations are then isolated through culturing, filtering and magnetic separation using cell-surface antigen markers. Here we provide a comprehensive protocol on how to isolate and culture individual cell types from whole endometrial tissues for use in in vitro experiments.

Isolation of Highly Pure Primary Mouse Alveolar Epithelial Type II Cells by Flow Cytometric Cell Sorting

Featured protocol,  Authors: Meenal Sinha
Meenal SinhaAffiliation: Department of Laboratory Medicine and the Program in Immunology, University of California, San Francisco, USA
For correspondence: meenal.sinha@ucsf.edu
Bio-protocol author page: a3723
 and Clifford A. Lowell
Clifford A. LowellAffiliation: Department of Laboratory Medicine and the Program in Immunology, University of California, San Francisco, USA
Bio-protocol author page: a3724
date: 11/20/2016, 119 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2013.

Brief version appeared in AJRCMB, Jun 2016
In this protocol, we describe the method for isolating highly pure primary alveolar epithelial type II (ATII) cells from lungs of naïve mice. The method combines negative selection for a variety of lineage markers along with positive selection for EpCAM, a pan-epithelial cell marker. This method yields 2-3 x 106 ATII cells per mouse lung. The cell preps are highly pure and viable and can be used for genomic or proteomic analyses or cultured ex vivo to understand their roles in various biological processes.

Documentation of floral secretory glands in Pleurothallidinae (Orchidaceae) using Scanning Electron Microscopy (SEM)

Featured protocol,  Authors: Adam P. Karremans
Adam P. KarremansAffiliation 1: Lankester Botanical Garden, University of Costa Rica, Cartago, Costa Rica
Affiliation 2: Naturalis Biodiversity Center, Leiden, The Netherlands
For correspondence: adam.karremans@ucr.ac.cr
Bio-protocol author page: a3744
Bertie Joan van Heuven
Bertie Joan van HeuvenAffiliation: Naturalis Biodiversity Center, Leiden, The Netherlands
Bio-protocol author page: a3745
Rob Langelaan
Rob LangelaanAffiliation: Naturalis Biodiversity Center, Leiden, The Netherlands
Bio-protocol author page: a3746
 and Barbara Gravendeel
Barbara GravendeelAffiliation: Naturalis Biodiversity Center, Leiden, The Netherlands
Bio-protocol author page: a3747
date: 11/20/2016, 105 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2021.

Brief version appeared in Ann Bot, Sep 2015
A clear, step by step description of the treatment of orchid flowers, subtribe Pleurothallidinae, with Critical Point Drying for SEM is presented. It shows that a simple, short fixation and dehydration method prior to Critical Point Drying is sufficient to obtain good results.

Uptake Assay for Radiolabeled Peptides in Yeast

Featured protocol,  Authors: Melinda Hauser
Melinda HauserAffiliation: Department of Microbiology, University of Tennessee, Knoxville, USA
Bio-protocol author page: a3756
Houjian Cai
Houjian CaiAffiliation: Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, University of Georgia, Athens, USA
Bio-protocol author page: a3755
Fred Naider
Fred NaiderAffiliation 1: Department of Chemistry and Macromolecular Assembly Institute, College of Staten Island of the City University of New York, Staten Island, New York, USA
Affiliation 2: Ph.D. Programs in Biochemistry and Chemistry, The Graduate Center of the City University of New York, New York, USA
Bio-protocol author page: a3757
 and Jeffrey M. Becker
Jeffrey M. BeckerAffiliation: Department of Microbiology, University of Tennessee, Knoxville, USA
For correspondence: jbecker@utk.edu
Bio-protocol author page: a3758
date: 11/20/2016, 117 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2026.

Brief version appeared in Eukaryot Cell, Oct 2007
We describe an assay for measuring the uptake of radioactive peptides into the yeast Saccharomyces cerevisiae. The methods presented here can be adapted to measure a variety of substrates transported into any bacterial or fungal cell via specific carrier-mediated systems.

Halo Assay for Toxic Peptides and Other Compounds in Microorganisms

Featured protocol,  Authors: Houjian Cai
Houjian CaiAffiliation: Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, University of Georgia, Athens, USA
Bio-protocol author page: a3755
Melinda Hauser
Melinda HauserAffiliation: Department of Microbiology, University of Tennessee, Knoxville, USA
Bio-protocol author page: a3756
Fred Naider
Fred NaiderAffiliation 1: Department of Chemistry and Macromolecular Assembly Institute, College of Staten Island of the City University of New York, Staten Island, USA
Affiliation 2: Ph.D. Programs in Biochemistry and Chemistry, The Graduate Center of the City University of New York, New York, USA
Bio-protocol author page: a3757
 and Jeffrey M. Becker
Jeffrey M. BeckerAffiliation: Department of Microbiology, University of Tennessee, Knoxville, USA
For correspondence: jbecker@utk.edu
Bio-protocol author page: a3758
date: 11/20/2016, 122 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2025.

Brief version appeared in Eukaryot Cell, Oct 2007
We describe an assay for determination of toxicity in S. cerevisiae involving spotting of a toxic peptide on a lawn of yeast cells. This assay may be generalized to determine toxicity of a variety of compounds by substituting a putative toxic compound in place of the peptide. The general protocol may also be used to determine toxicity of any small compound toward another microorganism by replacing S. cerevisiae with the target microbe and modifying growth conditions accordingly.

Determining the Influence of Small Molecules on Hypoxic Prostate Cancer Cell (DU-145) Viability Using Automated Cell Counting and a Cell Harvesting Protocol

Featured protocol,  Authors: John P Phelan
John P PhelanAffiliation: Biomerit Research Centre, School of Microbiology, University College Cork, Cork, Ireland
Bio-protocol author page: a3733
F Jerry Reen
F Jerry ReenAffiliation: Biomerit Research Centre, School of Microbiology, University College Cork, Cork, Ireland
Bio-protocol author page: a3775
 and Fergal O’Gara
Fergal O’GaraAffiliation 1: Biomerit Research Centre, School of Microbiology, University College Cork, Cork, Ireland
Affiliation 2: School of Biomedical Sciences, Curtin University, Perth, Australia
For correspondence: f.ogara@ucc.ie
Bio-protocol author page: a3734
date: 11/20/2016, 109 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2017.

Brief version appeared in BMC Cancer, Jul 2016
Cell viability assays are an essential aspect of most cancer studies, however they usually require a considerable labor and time input. Here, instead of using the conventional microscopy and hemocytometer cell counting approach, we developed a cell harvesting protocol and combined it with the automated Countess Automated Cell Counter to generate cell viability data. We investigated the effects of dihydroxylated bile acids on the cell viability of prostate cancer cells grown under hypoxic conditions. We observed that for all conditions, cell viability was relatively unchanged, suggesting these molecules had little or no impact on cell viability. The combination of the automated approach and the cell harvesting protocol means this assay is i) easy to perform, ii) extremely reproducible and iii) it complements more conventional cancer assay data such as invasion, migration and adhesion.

Determination of Rate of [3H-methyl]-choline Incorporation into Cellular Lipids and Non-lipid Metabolites

Featured protocol,  Authors: Tim Andrew Davies Smith
Tim Andrew Davies SmithAffiliation: School of Medicine, Medical Sciences and Nutrition, University of Aberdeen, Foresterhill, Aberdeen, UK
For correspondence: t.smith@abdn.ac.uk
Bio-protocol author page: a3739
 and Su Myat Phyu
Su Myat PhyuAffiliation: School of Medicine, Medical Sciences and Nutrition, University of Aberdeen, Foresterhill, Aberdeen, UK
Bio-protocol author page: a3740
date: 11/20/2016, 113 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2019.

Brief version appeared in PLoS One, Mar 2016
The choline-containing phospholipid, phosphatidylcholine (PtdCho) is the most common mammalian phospholipid found in cell membrane (Ide et al., 2013). It is also a component of intracellular signalling pathways (Cui and Houweling, 2002). Herein is described a measure of the rate of accumulation of choline by lipid soluble PtdCho and lyso-Ptdcho which can further be discriminated by chromatographic analysis (Smith and Phyu, 2016). Determination of the accumulation of [3H-methyl]-choline into water-soluble components is also described. The procedure could be used to measure the effect of drugs and other factors on choline incorporation into phospholipids. After exposure of cells to test conditions (e.g., drugs) adherent cells in tissue culture flasks are incubated with radiolabelled [3H-methyl]-choline in medium for 15 min (pulse). The [3H-methyl]-choline is then rapidly removed and incubation continued in the presence of non-radioactive medium (chase). Cellular distribution of [3H-methyl] is then determined by cell fractionation and measurement of radioactivity in the lipid and non-lipid cellular components.

Isolation of Latex Bead Phagosomes from Dictyostelium for in vitro Functional Assays

Authors: Ashwin D’Souza
Ashwin D’SouzaAffiliation: Department of Biological Sciences, Tata Institute of Fundamental Research, Mumbai, India
Bio-protocol author page: a3850
Paulomi Sanghavi
Paulomi SanghaviAffiliation: Department of Biological Sciences, Tata Institute of Fundamental Research, Mumbai, India
Bio-protocol author page: a3851
Ashim Rai
Ashim RaiAffiliation: Department of Biological Sciences, Tata Institute of Fundamental Research, Mumbai, India
Bio-protocol author page: a3852
Divya Pathak
Divya PathakAffiliation: Department of Biological Sciences, Tata Institute of Fundamental Research, Mumbai, India
Bio-protocol author page: a3853
 and Roop Mallik
Roop MallikAffiliation: Department of Biological Sciences, Tata Institute of Fundamental Research, Mumbai, India
For correspondence: roop@tifr.res.in
Bio-protocol author page: a3854
date: 12/5/2016, 73 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2056.

[Abstract] We describe a protocol to purify latex bead phagosomes (LBPs) from Dictyostelium cells. These can be later used for various in vitro functional assays. For instance, we use these LBPs to understand the microtubule motor-driven transport on in vitro polymerized microtubules. Phagosomes are allowed to mature for defined periods inside cells before extraction ...

Lymphocyte Isolation, Th17 Cell Differentiation, Activation, and Staining

Authors: Pawan Kumar
Pawan KumarAffiliation: Richard King Mellon Foundation Institute for Pediatric Research, Children’s Hospital of Pittsburgh of UPMC, University of Pittsburgh, Pittsburgh, PA, USA
For correspondence: pawan.kumar3@chp.edu
Bio-protocol author page: a2949
 and Jay K Kolls
Jay K KollsAffiliation: Richard King Mellon Foundation Institute for Pediatric Research, Children’s Hospital of Pittsburgh of UPMC, University of Pittsburgh, Pittsburgh, PA, USA
For correspondence: Jay.kolls@chp.edu
Bio-protocol author page: a3831
date: 12/5/2016, 35 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2047.

[Abstract] In vitro Th17 (α, β T helper cell which produce IL-17A, IL-17F and IL-22) differentiation has been routinely used for functional T cells studies. Here we describe a method for Th17 cell differentiation.
Keywords: Th17, IL-17, FACS

[Background] T cells are critical to mediate host defense against bacteria, viruses and fungi as well as commensal (Kumar ...


Vascular Smooth Muscle Cell Isolation and Culture from Mouse Aorta

Authors: Callie S. Kwartler
Callie S. KwartlerAffiliation: Department of Internal Medicine, Division of Medical Genetics, University of Texas Health Science Center, Houston, Texas, USA
Bio-protocol author page: a3821
Ping Zhou
Ping ZhouAffiliation: Department of Internal Medicine, Division of Medical Genetics, University of Texas Health Science Center, Houston, Texas, USA
Bio-protocol author page: a3822
Shao-Qing Kuang
Shao-Qing KuangAffiliation: Department of Internal Medicine, Division of Medical Genetics, University of Texas Health Science Center, Houston, Texas, USA
Bio-protocol author page: a3823
Xue-Yan Duan
Xue-Yan DuanAffiliation: Department of Internal Medicine, Division of Medical Genetics, University of Texas Health Science Center, Houston, Texas, USA
Bio-protocol author page: a3824
Limin Gong
Limin GongAffiliation: Department of Internal Medicine, Division of Medical Genetics, University of Texas Health Science Center, Houston, Texas, USA
Bio-protocol author page: a3825
 and Dianna M. Milewicz
Dianna M. MilewiczAffiliation: Department of Internal Medicine, Division of Medical Genetics, University of Texas Health Science Center, Houston, Texas, USA
For correspondence: Dianna.M.Milewicz@uth.tmc.edu
Bio-protocol author page: a3827
date: 12/5/2016, 29 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2045.

[Abstract] Vascular smooth muscle cells (SMC) in the ascending thoracic aorta arise from neural crest cells, whereas SMCs in the descending aorta are derived from the presomitic mesoderm. SMCs play important roles in cardiovascular development and aortic aneurysm formation. This protocol describes the detailed process for explanting ascending and descending SMCs ...

Analysis of Myosin II Minifilament Orientation at Epithelial Zonula Adherens

Authors: Magdalene Michael
Magdalene MichaelAffiliation: Randall Division of Cell and Molecular Biophysics, King’s College London, Guy’s Campus, London, UK
For correspondence: magdalene.michael@kcl.ac.uk
Bio-protocol author page: a3844
Xuan Liang
Xuan LiangAffiliation: Divisions of Cell Biology and Molecular Medicine, Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Brisbane, Queensland, Australia
Bio-protocol author page: a3845
 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, Queensland, Australia
For correspondence: g.gomez@uq.edu.au
Bio-protocol author page: a910
date: 12/5/2016, 37 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2054.

[Abstract] Non-muscle myosin II (NMII) form bipolar filaments, which bind F-actin to exert cellular contractility during physiological processes (Vicente-Manzanares et al., 2009). Using a combinatorial approach to fluorescently label both N- and C-termini of the NMII heavy chain, recent works have demonstrated the ability to visualize NMII bipolar filaments at ...

Lentiviral shRNA Screen to Identify Epithelial Integrity Regulating Genes in MCF10A 3D Culture

Authors: Elsa Marques
Elsa Marques Affiliation: Cancer Cell Circuitry Laboratory, Research Programs Unit/Translational Cancer Biology, University of Helsinki, Helsinki, Finland
Bio-protocol author page: a3834
 and Juha Klefström
Juha KlefströmAffiliation: Cancer Cell Circuitry Laboratory, Research Programs Unit/Translational Cancer Biology, University of Helsinki, Helsinki, Finland
For correspondence: juha.klefstrom@helsinki.fi
Bio-protocol author page: a3835
date: 12/5/2016, 71 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2050.

[Abstract] MCF10A 3D culture system provides a reductionist model of glandular mammary epithelium which is widely used to study development of glandular architecture, the role of cell polarity and epithelial integrity in control of epithelial cell functions, and mechanisms of breast cancer. Here we describe how to use shRNA screening approach to identify critical ...

DNA Damage Induction by Laser Microirradiation

Authors: Marianna Tampere
Marianna TampereAffiliation: Science for Life Laboratory, Division of Translational Medicine and Chemical Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden
Bio-protocol author page: a3807
 and Oliver Mortusewicz
Oliver MortusewiczAffiliation: Science for Life Laboratory, Division of Translational Medicine and Chemical Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden
For correspondence: oliver.mortusewicz@scilifelab.se
Bio-protocol author page: a3808
date: 12/5/2016, 46 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2039.

[Abstract] Genome instability can lead to cell death, senescence and cancerous transformation. Specific repair pathways have evolved to prevent accumulation of DNA lesions. Studying these highly dynamic and specific repair pathways requires precise spatial and temporal resolution, which can be achieved through a combination of laser microirradiaiton and live ...

In vitro Chondrogenic Hypertrophy Induction of Mesenchymal Stem Cells

Authors: Sang Young Jeong
Sang Young JeongAffiliation: Biomedical Research Institute, R&D Center, MEDIPOST Co., Ltd., Gyeonggi-do, Republic of Korea
Bio-protocol author page: a3855
Miyoung Lee
Miyoung LeeAffiliation: Biomedical Research Institute, R&D Center, MEDIPOST Co., Ltd., Gyeonggi-do, Republic of Korea
Bio-protocol author page: a3856
Soo Jin Choi
Soo Jin ChoiAffiliation: Biomedical Research Institute, R&D Center, MEDIPOST Co., Ltd., Gyeonggi-do, Republic of Korea
Bio-protocol author page: a3857
Wonil Oh
Wonil OhAffiliation: Biomedical Research Institute, R&D Center, MEDIPOST Co., Ltd., Gyeonggi-do, Republic of Korea
Bio-protocol author page: a3858
 and Hong Bae Jeon
Hong Bae JeonAffiliation: Biomedical Research Institute, R&D Center, MEDIPOST Co., Ltd., Gyeonggi-do, Republic of Korea
For correspondence: jhb@medi-post.co.kr
Bio-protocol author page: a3859
date: 12/5/2016, 42 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2057.

[Abstract] To investigate underlying mechanism of chondrogenic hypertrophy, we need proper in vitro hypertrophic model of mesenchymal stem cells (MSCs). This protocol describes our defined method for induction of in vitro chondrogenic hypertrophy of human umbilical cord blood-derived MSCs (hUCB-MSCs). By adding thyroid hormone (T3; triiodothyronine) and minimum ...

Mouse Model of Dengue Virus Infection with Serotypes 1 and 2 Clinical Isolates

Authors: Satoru Watanabe
Satoru Watanabe Affiliation: Emerging Infectious Diseases, Duke-NUS Medical School, Singapore, Singapore
For correspondence: satoru.watanabe@duke-nus.edu.sg
Bio-protocol author page: a3270
Kitti Wing Ki Chan
Kitti Wing Ki ChanAffiliation: Emerging Infectious Diseases, Duke-NUS Medical School, Singapore, Singapore
Bio-protocol author page: a3810
 and Subhash G. Vasudevan
Subhash G. VasudevanAffiliation: Emerging Infectious Diseases, Duke-NUS Medical School, Singapore, Singapore
For correspondence: subhash.vasudevan@duke-nus.edu.sg
Bio-protocol author page: a3811
date: 12/5/2016, 33 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2040.

[Abstract] Dengue is a global public health threat caused by infection with any of the 4 related dengue virus serotypes (DENV1-4). Clinical manifestations range from self-limiting febrile illness, known as dengue fever (DF), to life-threatening severe diseases, such as dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS). Most cases of DHF/DSS are associated ...

Measurement of Intracellular Calcium Concentration in Pseudomonas aeruginosa

Authors: Manita Guragain
Manita GuragainAffiliation: Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, US
Bio-protocol author page: a3812
Anthony K. Campbell
Anthony K. CampbellAffiliation: Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, US
Bio-protocol author page: a3813
 and Marianna A. Patrauchan
Marianna A. PatrauchanAffiliation: Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, US
For correspondence: m.patrauchan@okstate.edu
Bio-protocol author page: a3814
date: 12/5/2016, 31 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2041.

[Abstract] Characterization of the molecular mechanisms of calcium (Ca2+) regulation of bacterial physiology and virulence requires tools enabling measuring and monitoring the intracellular levels of free calcium (Ca2+in). Here, we describe a protocol optimized to use a recombinantly expressed Ca2+-binding protein, aequorin, for detecting Ca2+in in Pseudomonas ...

Various Modes of Spinal Cord Injury to Study Regeneration in Adult Zebrafish

Authors: Subhra Prakash Hui
Subhra Prakash HuiAffiliation 1: Department of Biophysics, Molecular Biology and Bioinformatics, University of Calcutta, Kolkata, India
Affiliation 2: Victor Chang Cardiac Research Institute, Lowy Packer Building, Darlinghurst, Australia
Bio-protocol author page: a3817
 and Sukla Ghosh
Sukla GhoshAffiliation: Department of Biophysics, Molecular Biology and Bioinformatics, University of Calcutta, Kolkata, India
For correspondence: suklagh2010@gmail.com
Bio-protocol author page: a3818
date: 12/5/2016, 42 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2043.

[Abstract] Spinal cord injury (SCI) in mammals leads to failure of both sensory and motor functions, due to lack of axonal regrowth below the level of injury as well as inability to replace lost neural cells and to stimulate neurogenesis. In contrast, fish and amphibians are capable of regenerating a variety of their organs like limb/fin, jaw, heart and various ...
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 

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, 50813 views, 6 Q&A, How to cite
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, 41083 views, 7 Q&A, How to cite
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, 36844 views, 2 Q&A, How to cite
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, 36669 views, 2 Q&A, How to cite
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 ...

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, 35698 views, 4 Q&A, How to cite
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 ...

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, 34055 views, 5 Q&A, How to cite
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, 33592 views, 15 Q&A, How to cite
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, 30116 views, 1 Q&A, How to cite
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, 29540 views, 6 Q&A, How to cite
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] Calcium Phosphate Transfection of Eukaryotic Cells

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: 2/5/2012, 25545 views, 2 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.86.

[Abstract] Transfection of DNA into cells is an indispensible protocol in molecular biology. While plenty of lipid-based transfection reagents are commercially available nowadays, a quick, simple, efficient and inexpensive method is to transfect eukaryotic cells via calcium phosphate co-precipitation with DNA ...
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55