Welcome guest, Sign in

Home

Mammalia

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.

Preparation of Purified Gram-positive Bacterial Cell Wall and Detection in Placenta and Fetal Tissues

Featured protocol,  Authors: Beth Mann
Beth MannAffiliation: Department of Infectious Diseases, St Jude Children’s Research Hospital, Memphis TN, USA
Bio-protocol author page: a3796
Lip Nam Loh
Lip Nam LohAffiliation: Department of Infectious Diseases, St Jude Children’s Research Hospital, Memphis TN, USA
Bio-protocol author page: a3797
Geli Gao
Geli GaoAffiliation: Department of Infectious Diseases, St Jude Children’s Research Hospital, Memphis TN, USA
Bio-protocol author page: a3798
 and Elaine Tuomanen
Elaine TuomanenAffiliation: Department of Infectious Diseases, St Jude Children’s Research Hospital, Memphis TN, USA
For correspondence: Elaine.tuomanen@stjude.org
Bio-protocol author page: a3799
date: 12/5/2016, 34 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2037.

Brief version appeared in Cell Host Microbe, Mar 2016
Cell wall is a complex biopolymer on the surface of all Gram-positive bacteria. During infection, cell wall is recognized by the innate immune receptor Toll-like receptor 2 causing intense inflammation and tissue damage. In animal models, cell wall traffics from the blood stream to many organs in the body, including brain, heart, placenta and fetus. This protocol describes how to prepare purified cell wall from Streptococcus pneumoniae, detect its distribution in animal tissues, and study the tissue response using the placenta and fetal brain as examples.

Murine Leukemia Virus (MLV)-based Coronavirus Spike-pseudotyped Particle Production and Infection

Featured protocol,  Authors: Jean Kaoru Millet
Jean Kaoru MilletAffiliation: Department of Microbiology and Immunology, Cornell University, Ithaca NY, United States
For correspondence: jkm248@cornell.edu
Bio-protocol author page: a3793
 and Gary R. Whittaker
Gary R. WhittakerAffiliation: Department of Microbiology and Immunology, Cornell University, Ithaca NY, United States
For correspondence: gary.whittaker@cornell.edu
Bio-protocol author page: a942
date: 12/5/2016, 32 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2035.

Brief version appeared in PNAS, Oct 2014
Viral pseudotyped particles (pp) are enveloped virus particles, typically derived from retroviruses or rhabdoviruses, that harbor heterologous envelope glycoproteins on their surface and a genome lacking essential genes. These synthetic viral particles are safer surrogates of native viruses and acquire the tropism and host entry pathway characteristics governed by the heterologous envelope glycoprotein used. They have proven to be very useful tools used in research with many applications, such as enabling the study of entry pathways of enveloped viruses and to generate effective gene-delivery vectors. The basis for their generation lies in the capacity of some viruses, such as murine leukemia virus (MLV), to incorporate envelope glycoproteins of other viruses into a pseudotyped virus particle. These can be engineered to contain reporter genes such as luciferase, enabling quantification of virus entry events upon pseudotyped particle infection with susceptible cells. Here, we detail a protocol enabling generation of MLV-based pseudotyped particles, using the Middle East respiratory syndrome coronavirus (MERS-CoV) spike (S) as an example of a heterologous envelope glycoprotein to be incorporated. We also describe how these particles are used to infect susceptible cells and to perform a quantitative infectivity readout by a luciferase assay.

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.

MPM-2 Mediated Immunoprecipitation of Proteins Undergoing Proline-directed Phosphorylation

Featured protocol,  Authors: Roberta Antonelli
Roberta AntonelliAffiliation 1: International School for Advanced Studies, Neurobiology Department, Trieste, Italy
Affiliation 2: Laboratory of Translational Research in Child and Adolescent Cancer, Vall d'Hebron Research Institute (VHIR)-UAB, Barcelona, Spain
For correspondence: roberta.antonelli@vhir.org
Bio-protocol author page: a3828
 and Paola Zacchi
Paola ZacchiAffiliation: International School for Advanced Studies, Neurobiology Department, Trieste, Italy
Bio-protocol author page: a3829
date: 12/5/2016, 26 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2046.

Brief version appeared in J Neurosci, May 2016
Immunoprecipitation (IP) represents a widely utilized biochemical method to isolate a specific protein from a complex mixture taking advantage of an antibody that specifically recognizes that particular target molecule. This procedure is extremely versatile and can be applied to concentrate a specific protein, to identify interacting partners in complex with it or to detect post-translational modifications. The mitotic protein monoclonal 2 (MPM-2) is an antibody originally raised against extracts of synchronized mitotic HeLa cells to identify proteins selectively present in mitotic, and not in interphase-cells (Davis et al., 1983). MPM-2 recognizes phosphorylated serine or threonine residues followed by proline (pS/T-P), consensus epitopes generated by the concerted action of proline-directed protein kinases and phosphatases (Lu et al., 2002). These reversible phosphorylation events have emerged to control various cellular processes by promoting conformational changes on the target that are not simply due to the phosphorylation event per se. These motifs, once phosphorylated, are able to recruit Pin1 (Peptidyl-prolyl Isomerase NIMA interacting protein 1) (Lu et al., 1996; Lu and Zhou, 2007), a chaperone which drives the cis/trans isomerization reaction on the peptide bond, switching the substrate between functionally diverse conformations (Lu, 2004; Wulf et al., 2005). This protocol describes a general MPM-2 based immunoprecipitation strategy using the scaffolding molecule postsynaptic density protein-95 (PSD-95) (Chen et al., 2005), a neuronal Pin1 target (Antonelli et al., 2016), as an example to illustrate the detailed procedure.

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.

Delayed Spatial Win-shift Test on Radial Arm Maze

Featured protocol,  Authors: Simone N. De Luca
Simone N. De LucaAffiliation: School of Health and Biomedical Sciences, RMIT University, Melbourne, Vic, Australia
Bio-protocol author page: a3841
Luba Sominsky
Luba Sominsky Affiliation: School of Health and Biomedical Sciences, RMIT University, Melbourne, Vic, Australia
Bio-protocol author page: a3842
 and Sarah J. Spencer
Sarah J. SpencerAffiliation: School of Health and Biomedical Sciences, RMIT University, Melbourne, Vic, Australia
For correspondence: Sarah.Spencer@rmit.edu.au
Bio-protocol author page: a3843
date: 12/5/2016, 29 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2053.

Brief version appeared in J Neuroinflammation, May 2016
The radial arm maze (RAM) is used to assess reference and working memory in rodents. This task relies on the rodent’s ability to orientate itself in the maze using extra-maze visual cues. This test can be used to investigate whether a rodent’s cognition is improved or impaired under a variety of experimental conditions. Here, we describe one way to test spatial working and reference memory. This delayed spatial win-shift (DSWS) procedure on the RAM was adapted from Packard and White (1990). The win-shift component of the test refers to the alternation of baiting, or rewarding, arms during the trial and test phase. The rodent is required to hold spatial information both within the task and across a delay to obtain the food-pellet reward (Taylor et al., 2003b). This task measures the incidence and type of memory errors made by the rodent both in the training and test phases of the learning task. A working memory error (re-entry of an arm that has been baited) can occur in both phases of the task, whilst a reference memory error (entry into an arm that has been baited during the training phase and is no longer baited) can only occur during the test phase.

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.

Assay to Evaluate BAL Fluid Regulation of Fibroblast α-SMA Expression

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, 152 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2009.

Brief version appeared in Immunity, Mar 2016
Because transforming growth factor-β (TGF-β1) induces differentiation of fibroblasts to myofibroblasts, we developed a protocol to evaluate alveolar macrophage-derived TGF-β1 regulation of lung fibroblast differentiation (Larson-Casey et al., 2016). The protocol evaluates the ability of mouse bronchoalveolar lavage (BAL) fluid to alter fibroblast differentiation. Fibroblast differentiation was measured by the expression of α-smooth muscle actin (α-SMA).

Establishment of Patient-Derived Xenografts in Mice

Featured protocol,  Authors: Dongkyoo Park
Dongkyoo ParkAffiliation: Division of Cancer Biology, Department of Radiation Oncology, Emory University School of Medicine and Winship Cancer Institute of Emory University, Atlanta, GA, USA
Bio-protocol author page: a3712
Dongsheng Wang
Dongsheng WangAffiliation: Division of Cancer Biology, Department of Radiation Oncology, Emory University School of Medicine and Winship Cancer Institute of Emory University, Atlanta, GA, USA
Bio-protocol author page: a3713
Guo Chen
Guo ChenAffiliation: Division of Cancer Biology, Department of Radiation Oncology, Emory University School of Medicine and Winship Cancer Institute of Emory University, Atlanta, GA, USA
Bio-protocol author page: a3714
 and Xingming Deng
Xingming DengAffiliation: Division of Cancer Biology, Department of Radiation Oncology, Emory University School of Medicine and Winship Cancer Institute of Emory University, Atlanta, GA, USA
For correspondence: xdeng4@emory.edu
Bio-protocol author page: a3715
date: 11/20/2016, 189 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2008.

Brief version appeared in Cancer Cell, Jun 2015
Patient-derived xenograft (PDX) models for cancer research have recently attracted considerable attention in both the academy and industry (Hidalgo et al., 2014; Wilding and Bodmer, 2014). PDX models have been developed from different tumor types including lung cancer to improve the drug development process. These models are used for pre-clinical drug evaluation and can be used for the predictive results of clinical outcomes because they conserve original tumor characteristics such as heterogeneity, complexity and molecular diversity (Kopetz et al., 2012). Additionally, PDX model provides the potential tool for the personalized drug therapy. In this protocol, we present methods for the establishment of PDX in mice using primary tumor tissues from patients with small cell lung cancer (SCLC).

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.

Total Histone Acid Extraction of Colon Cancer HCT116 Cells

Featured protocol,  Authors: Lin-Lin Cao
Lin-Lin CaoAffiliation: Department of Clinical Laboratory, Peking University People’s Hospital, Beijing, China
Bio-protocol author page: a3751
 and Wei-Guo Zhu
Wei-Guo ZhuAffiliation 1: Department of Biochemistry and Molecular Biology, School of Medicine, Shenzhen University, Shenzhen, China
Affiliation 2: Peking University-Tsinghua University Joint Center for Life Sciences, Beijing, China
For correspondence: zhuweiguo@szu.edu.cn
Bio-protocol author page: a3752
date: 11/20/2016, 117 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2023.

Brief version appeared in Oncogene, Jan 2016
Histone acid extraction assay is a popular method to determine histone modification levels in mammalian cells. It includes three steps: first, histones are released from chromatin by sulfuric acid; trichloroacetate (TCA) is then added to precipitate histones; and finally, histones are dissolved in double-distilled H2O (ddH2O). Here we present a detailed histone acid extraction assay in our laboratory using a colon cancer cell line, HCT116, as a model.

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.

In vitro Brainstem-spinal Cord Preparation from Newborn Rat

Featured protocol,  Authors: Jean-Patrick Le Gal
Jean-Patrick Le GalAffiliation: Institut de Neurosciences Cognitives et Intégratives d’Aquitaine (INCIA), Université de Bordeaux, Bordeaux, France
Bio-protocol author page: a3699
Angelo Nicolosi
Angelo NicolosiAffiliation: Institut de Neurosciences Cognitives et Intégratives d’Aquitaine (INCIA), Université de Bordeaux, Bordeaux, France
Bio-protocol author page: a3700
Laurent Juvin
Laurent JuvinAffiliation: Institut de Neurosciences Cognitives et Intégratives d’Aquitaine (INCIA), Université de Bordeaux, Bordeaux, France
Bio-protocol author page: a3701
 and Didier Morin
Didier MorinAffiliation: Institut de Neurosciences Cognitives et Intégratives d’Aquitaine (INCIA), Université de Bordeaux, Bordeaux, France
For correspondence: didier.morin@u-bordeaux.fr
Bio-protocol author page: a3702
date: 11/20/2016, 111 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2003.

Brief version appeared in J Neurosci, Jan 2016
The brainstem-spinal cord preparation of newborn rat contains neural networks able to produce motor output in absence of sensory feedback. These neural structures, commonly called central pattern generators (CPGs), are involved in many vital functions such as respiration (Morin and Viala, 2002; Giraudin et al., 2008) or locomotion (Juvin et al., 2005). Here we describe a procedure for the isolation of the brainstem-spinal cord tissue of neonatal rat (0-2 days old). A surgical method under binocular microscope allows the brainstem and the spinal cord to be isolated in vitro and the motor outputs to be recorded. This preparation can then be used for diverse experimental approaches, such as electrophysiology, pharmacology or anatomical studies, and constitutes a useful model to study the interaction between CPGs (Juvin et al., 2007; 2012; Giraudin et al., 2012; Le Gal et al., 2014; 2016).

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.

Biochemical Analysis of Caspase-8-dependent Proteolysis of IRF3 in Virus-infected Cells

Featured protocol,  Authors: Gayatri Subramanian
Gayatri SubramanianAffiliation: Department of Medical Microbiology and Immunology, University of Toledo College of Medicine and Life Sciences, Toledo, USA
Bio-protocol author page: a3735
Karen Pan
Karen PanAffiliation: Department of Medical Microbiology and Immunology, University of Toledo College of Medicine and Life Sciences, Toledo, USA
Bio-protocol author page: a3736
Ritu Chakravarti
Ritu ChakravartiAffiliation: Department of Surgery, University of Toledo College of Medicine and Life Sciences, Toledo, USA
Bio-protocol author page: a3737
 and Saurabh Chattopadhyay
Saurabh ChattopadhyayAffiliation: Department of Medical Microbiology and Immunology, University of Toledo College of Medicine and Life Sciences, Toledo, USA
For correspondence: Saurabh.Chattopadhyay@UToledo.edu
Bio-protocol author page: a3738
date: 11/20/2016, 145 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2018.

Brief version appeared in J Biol Chem, Sep 2011
Interferon regulatory factor 3 (IRF3) is a transcription factor, which is critical for the antiviral response against a wide range of viruses (Hiscott, 2007; Ikushima et al., 2013). It gets activated in virus-infected cells via Toll like receptors (TLRs), RIG-I (retinoic acid inducible gene 1) like receptors (RLRs), cyclic GMP-AMP synthase (cGAS) – stimulator of interferon genes (STING), which are sensors of viral components in the cells (Chattopadhyay and Sen, 2014a; 2014b; Hiscott, 2007). IRF3 is a cytoplasmic protein, upon activation by virally activated sensors it gets phosphorylated, translocated to the nucleus and binds to the interferon-sensitive response element (ISRE) of the gene promoters to induce their transcription (Hiscott, 2007). IRF3 has other functions, including direct stimulation of apoptosis in virus-infected cells. In this pathway, the transcriptional activity of IRF3 is not required (Chattopadhyay et al., 2013b; Chattopadhyay et al., 2016; Chattopadhyay et al., 2010; Chattopadhyay and Sen, 2010; Chattopadhyay et al., 2011). These pathways are negatively regulated by host factors as well as by viruses. Our studies indicate that IRF3 can be proteolytically processed by caspase-8-dependent cleavage (Sears et al., 2011). A specific site in IRF3 is targeted by caspase-8, activated in RNA or DNA virus-infected and dsRNA-stimulated cells (Sears et al., 2011). The direct involvement of caspase-8 was confirmed by in vitro cleavage assay using recombinant proteins and in vivo by virus activated caspase-8. The proteolytic cleavage of IRF3 can be inhibited by chemical inhibition or genetic ablation of caspase-8. The cleavage of IRF3 removes the activated pool of IRF3 and thus can be used as a pro-viral mechanism (Figure 1). Using a C-terminally epitope-tagged human IRF3, we analyzed the cleavage of IRF3 in virus-infected cells. Moreover, we used recombinant proteins in vitro to conclude that IRF3 is a substrate of caspase-8 (Sears et al., 2011). In the current protocol, we have outlined a simple and detailed procedure to biochemically analyze the proteolysis of IRF3 in virus-infected cells and the specific role of caspase-8 in this process.

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.

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.

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, ...


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 ...

Preparation of Purified Gram-positive Bacterial Cell Wall and Detection in Placenta and Fetal Tissues

Authors: Beth Mann
Beth MannAffiliation: Department of Infectious Diseases, St Jude Children’s Research Hospital, Memphis TN, USA
Bio-protocol author page: a3796
Lip Nam Loh
Lip Nam LohAffiliation: Department of Infectious Diseases, St Jude Children’s Research Hospital, Memphis TN, USA
Bio-protocol author page: a3797
Geli Gao
Geli GaoAffiliation: Department of Infectious Diseases, St Jude Children’s Research Hospital, Memphis TN, USA
Bio-protocol author page: a3798
 and Elaine Tuomanen
Elaine TuomanenAffiliation: Department of Infectious Diseases, St Jude Children’s Research Hospital, Memphis TN, USA
For correspondence: Elaine.tuomanen@stjude.org
Bio-protocol author page: a3799
date: 12/5/2016, 34 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2037.

[Abstract] Cell wall is a complex biopolymer on the surface of all Gram-positive bacteria. During infection, cell wall is recognized by the innate immune receptor Toll-like receptor 2 causing intense inflammation and tissue damage. In animal models, cell wall traffics from the blood stream to many organs in the ...

Murine Leukemia Virus (MLV)-based Coronavirus Spike-pseudotyped Particle Production and Infection

Authors: Jean Kaoru Millet
Jean Kaoru MilletAffiliation: Department of Microbiology and Immunology, Cornell University, Ithaca NY, United States
For correspondence: jkm248@cornell.edu
Bio-protocol author page: a3793
 and Gary R. Whittaker
Gary R. WhittakerAffiliation: Department of Microbiology and Immunology, Cornell University, Ithaca NY, United States
For correspondence: gary.whittaker@cornell.edu
Bio-protocol author page: a942
date: 12/5/2016, 32 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2035.

[Abstract] Viral pseudotyped particles (pp) are enveloped virus particles, typically derived from retroviruses or rhabdoviruses, that harbor heterologous envelope glycoproteins on their surface and a genome lacking essential genes. These synthetic viral particles are safer surrogates of native viruses and acquire ...

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 ...

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 ...

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 ...

MPM-2 Mediated Immunoprecipitation of Proteins Undergoing Proline-directed Phosphorylation

Authors: Roberta Antonelli
Roberta AntonelliAffiliation 1: International School for Advanced Studies, Neurobiology Department, Trieste, Italy
Affiliation 2: Laboratory of Translational Research in Child and Adolescent Cancer, Vall d'Hebron Research Institute (VHIR)-UAB, Barcelona, Spain
For correspondence: roberta.antonelli@vhir.org
Bio-protocol author page: a3828
 and Paola Zacchi
Paola ZacchiAffiliation: International School for Advanced Studies, Neurobiology Department, Trieste, Italy
Bio-protocol author page: a3829
date: 12/5/2016, 26 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2046.

[Abstract] Immunoprecipitation (IP) represents a widely utilized biochemical method to isolate a specific protein from a complex mixture taking advantage of an antibody that specifically recognizes that particular target molecule. This procedure is extremely versatile and can be applied to concentrate a specific ...

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 ...

Delayed Spatial Win-shift Test on Radial Arm Maze

Authors: Simone N. De Luca
Simone N. De LucaAffiliation: School of Health and Biomedical Sciences, RMIT University, Melbourne, Vic, Australia
Bio-protocol author page: a3841
Luba Sominsky
Luba Sominsky Affiliation: School of Health and Biomedical Sciences, RMIT University, Melbourne, Vic, Australia
Bio-protocol author page: a3842
 and Sarah J. Spencer
Sarah J. SpencerAffiliation: School of Health and Biomedical Sciences, RMIT University, Melbourne, Vic, Australia
For correspondence: Sarah.Spencer@rmit.edu.au
Bio-protocol author page: a3843
date: 12/5/2016, 29 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2053.

[Abstract] The radial arm maze (RAM) is used to assess reference and working memory in rodents. This task relies on the rodent’s ability to orientate itself in the maze using extra-maze visual cues. This test can be used to investigate whether a rodent’s cognition is improved or impaired under a variety of experimental ...
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 56 57 58 59 60 61 62 63 64 65 66 67 

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] 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, 28746 views, 18 Q&A, How to cite
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 ...
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 56 57 58 59 60 61 62 63 64 65 66