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Differential Salt Fractionation of Nuclei to Analyze Chromatin-associated Proteins from Cultured Mammalian Cells

Featured protocol,  Authors: Christin Herrmann
Christin HerrmannAffiliation 1: Division of Cancer Pathobiology, Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia, Philadelphia, USA
Affiliation 2: Cell & Molecular Biology Graduate Group, University of Pennsylvania, Philadelphia, USA
Bio-protocol author page: a4254
Daphne C. Avgousti
Daphne C. AvgoustiAffiliation 1: Division of Cancer Pathobiology, Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia, Philadelphia, USA
Affiliation 2: Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, USA
Bio-protocol author page: a4255
 and Matthew D. Weitzman
Matthew D. WeitzmanAffiliation 1: Division of Cancer Pathobiology, Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia, Philadelphia, USA
Affiliation 2: Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, USA
For correspondence: weitzmanm@email.chop.edu
Bio-protocol author page: a4256
date: 3/20/2017, 123 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2175.

Brief version appeared in Nature, Jul 2016
Nucleosomes are the core units of cellular chromatin and are comprised of 147 base pairs (bp) of DNA wrapped around an octamer of histone proteins. Proteins such as chromatin remodelers, transcription factors, and DNA repair proteins interact dynamically with chromatin to regulate access to DNA, control gene transcription, and maintain genome integrity. The extent of association with chromatin changes rapidly in response to stresses, such as immune activation, oxidative stress, or viral infection, resulting in downstream effects on chromatin conformation and transcription of target genes. To elucidate changes in the composition of proteins associated with chromatin under different conditions, we adapted existing protocols to isolate nuclei and fractionate cellular chromatin using a gradient of salt concentrations. The presence of specific proteins in different salt fractions can be assessed by Western blotting or mass spectrometry, providing insight into the degree to which they are associated with chromatin.

MHC Class II Tetramer Labeling of Human Primary CD4+ T Cells from HIV Infected Patients

Featured protocol,  Authors: Moran Galperin
Moran GalperinAffiliation: Pasteur Institute, Viral Pathogenesis Unit, Paris, France
For correspondence: moran.galperin@pasteur.fr
Bio-protocol author page: a4268
Daniela Benati
Daniela BenatiAffiliation: Center for Regenerative Medicine “Stephano Ferrari”, Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy
Bio-protocol author page: a4269
Mathieu Claireaux
Mathieu ClaireauxAffiliation: Pasteur Institute, Viral Pathogenesis Unit, Paris, France
Bio-protocol author page: a4270
Madhura Mukhopadhyay
Madhura MukhopadhyayAffiliation: Pasteur Institute, Viral Pathogenesis Unit, Paris, France
Bio-protocol author page: a4271
 and Lisa A. Chakrabarti
Lisa A. ChakrabartiAffiliation: Pasteur Institute, Viral Pathogenesis Unit, Paris, France
Bio-protocol author page: a4272
date: 3/20/2017, 119 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2187.

Brief version appeared in J Clin Invest, Jun 2016
Major Histocompatibility Complex (MHC) tetramers have been used for two decades to detect, isolate and characterize T cells specific for various pathogens and tumor antigens. In the context of Human Immunodeficiency Virus (HIV) infection, antigen-specific CD8+ T cells have been extensively studied ex vivo, as they can be readily detected by HIV peptide-loaded MHC class I tetramers. In contrast, the detection of HIV-specific CD4+ T cells has proven more challenging, due to the intrinsically lower clonal expansion rates of CD4+ T cells, and to the preferential depletion of HIV-specific CD4+ T cells in the course of HIV infection.

Adoptive Transfer of Lung Antigen Presenting Cells

Featured protocol,  Authors: Xiaofeng Zhou
Xiaofeng ZhouAffiliation: Department of Internal Medicine, Pulmonary and Critical Care Medicine Division, University of Michigan, Ann Arbor, Michigan, USA
For correspondence: xiazhou@umich.edu
Bio-protocol author page: a4212
 and Bethany B Moore
Bethany B MooreAffiliation: Department of Internal Medicine, Pulmonary and Critical Care Medicine Division, University of Michigan, Ann Arbor, Michigan, USA
Bio-protocol author page: a4213
date: 3/20/2017, 92 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2182.

Brief version appeared in Mucosal Immunol, May 2016
Our protocol describes adoptive transfer of antigen presenting cells (APCs) isolated from the lungs by enzymatic digestion and magnetic enrichment. This protocol can be used to study APC functions and trafficking.

Mouse Model of Reversible Intestinal Inflammation

Featured protocol,  Authors: Cheong KC Kwong Chung
Cheong KC Kwong ChungAffiliation: Division of Experimental Pathology, Institute of Pathology, University of Bern, Bern, Switzerland
For correspondence: cheong.kwong@pathology.unibe.ch
Bio-protocol author page: a4194
Jennifer Brasseit
Jennifer BrasseitAffiliation: Division of Experimental Pathology, Institute of Pathology, University of Bern, Bern, Switzerland
Bio-protocol author page: a4190
Esther Althaus-Steiner
Esther Althaus-SteinerAffiliation: Division of Experimental Pathology, Institute of Pathology, University of Bern, Bern, Switzerland
Bio-protocol author page: a4191
Silvia Rihs
Silvia RihsAffiliation: Division of Experimental Pathology, Institute of Pathology, University of Bern, Bern, Switzerland
Bio-protocol author page: a4192
 and Christoph Mueller
Christoph MuellerAffiliation: Division of Experimental Pathology, Institute of Pathology, University of Bern, Bern, Switzerland
For correspondence: christoph.mueller@pathology.unibe.ch
Bio-protocol author page: a4193
date: 3/20/2017, 127 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2173.

Brief version appeared in Mucosal Immunol, May 2016
Current therapies to treat inflammatory bowel disease by dampening excessive inflammatory immune responses have had limited success (Reinisch et al., 2011; Rutgeerts et al., 2005; Sandborn et al., 2012). To develop new therapeutic interventions, there is a need for better understanding of the mechanisms that are operative during mucosal healing (Pineton de Chambrun et al., 2010). To this end, a reversible model of colitis was developed in which colitis induced by adoptive transfer of naïve CD4+ CD45RBhi T cells in lymphopenic mice can be reversed through depletion of colitogenic CD4+ T cells (Brasseit et al., 2016).

Determination of the Predatory Capability of Bdellovibrio bacteriovorus HD100

Featured protocol,  Authors: Cristina Herencias
Cristina HerenciasAffiliation: Environmental Biology Department, Centro de Investigaciones Biológicas, CSIC, Madrid, Spain
Bio-protocol author page: a4258
M. Auxiliadora Prieto
M. Auxiliadora PrietoAffiliation: Environmental Biology Department, Centro de Investigaciones Biológicas, CSIC, Madrid, Spain
Bio-protocol author page: a4259
 and Virginia Martínez
Virginia MartínezAffiliation: Environmental Biology Department, Centro de Investigaciones Biológicas, CSIC, Madrid, Spain
Present address: Evolva Biotech A/S, Copenhagen, Denmark
For correspondence: virginiaml83@gmail.com
Bio-protocol author page: a4257
date: 3/20/2017, 116 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2177.

Brief version appeared in Sci Rep, Apr 2016
Bdellovibrio bacteriovorus HD100 is an obligate predator that preys upon a wide variety of Gram negative bacteria. The biphasic growth cycle of Bdellovibrio includes a free-swimming attack phase and an intraperiplasmic growth phase, where the predator replicates its DNA and grows using the prey as a source of nutrients, finally dividing into individual cells (Sockett, 2009). Due to its obligatory predatory lifestyle, manipulation of Bdellovibrio requires two-member culturing techniques using selected prey microorganisms (Lambert et al., 2003). In this protocol, we describe a detailed workflow to grow and quantify B. bacteriovorus HD100 and its predatory ability, to easily carry out these laborious and time-consuming techniques.

In vitro Assay to Assess Efficacy of Potential Antiviral Compounds against Enterovirus D68

Featured protocol,  Authors: Liang Sun
Liang SunAffiliation: KU Leuven – University of Leuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, Leuven, Belgium
Bio-protocol author page: a4263
Leen Delang
Leen DelangAffiliation: KU Leuven – University of Leuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, Leuven, Belgium
Bio-protocol author page: a4264
Carmen Mirabelli
Carmen Mirabelli Affiliation: KU Leuven – University of Leuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, Leuven, Belgium
Bio-protocol author page: a4265
 and Johan Neyts
Johan NeytsAffiliation: KU Leuven – University of Leuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, Leuven, Belgium
For correspondence: johan.neyts@kuleuven.be
Bio-protocol author page: a1546
date: 3/20/2017, 85 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2183.

Brief version appeared in Antimicrob Agents Chemother, Dec 2015
In 2014 enterovirus D68 (EV-D68) caused the largest outbreak in the United States since the discovery of the virus. Distinct from before, the 2014 infections were associated with more severe respiratory disease and occasional neurological complications. So far, there are no available vaccines or antivirals for the prophylaxis or treatment of EV-D68 infections. In order to evaluate the antiviral activity of potential inhibitors of EV-D68 replication, a cell-based cytopathic effect (CPE) reduction assay was developed (Sun et al., 2015).

3D Stroma Invasion Assay

Featured protocol,  Authors: Yvette May Coulson-Thomas
Yvette May Coulson-ThomasAffiliation: Department of Biochemistry, Universidade Federal de São Paulo, São Paulo, Brazil
For correspondence: ycoulsonthomas@gmail.com
Bio-protocol author page: a4214
 and Vivien Jane Coulson-Thomas
Vivien Jane Coulson-ThomasAffiliation: College of Optometry, the Ocular Surface Institute (TOSI), University of Houston, Houston, USA
For correspondence: vcoulsonthomas@gmail.com
Bio-protocol author page: a1653
date: 3/20/2017, 99 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2195.

Brief version appeared in Cell Tissue Res, Nov 2011
We have developed a 3D co-culture system composed of fibroblasts and colorectal cancer cells that enables us to study the desmoplastic reaction. This method also enables us to study the influence of the desmoplastic reaction on the migration of colorectal cancer cells through the surrounding stroma. This protocol has been previously published (Coulson-Thomas et al., 2011) and is described here in more detail.

Reprogram Murine Epiblast Stem Cells by Epigenetic Inhibitors

Featured protocol,  Authors: Hui Zhang
Hui ZhangAffiliation: Department of Pathology, University of Michigan, Ann Arbor, USA
Bio-protocol author page: a4176
 and Yali Dou
Yali DouAffiliation: Department of Pathology, University of Michigan, Ann Arbor, USA
For correspondence: yalid@med.umich.edu
Bio-protocol author page: a4171
date: 3/5/2017, 170 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2168.

Brief version appeared in Cell Stem Cell, Apr 2016
Pluripotent stem cells in the naïve state are highly useful in regenerative medicine and tissue engineering. A robust reprogramming of the primed murine Epiblast Stem Cells (EpiSCs) to naïve pluripotency is feasible via chemical-only approach. This protocol described a method to reprogram murine EpiSCs by MM-401 treatment, which blocks histone H3K4 methylation by MLL1/KMT2A.

Ca2+ Measurements in Mammalian Cells with Aequorin-based Probes

Featured protocol,  Authors: Anna Tosatto
Anna TosattoAffiliation: Department of Biomedical Sciences, University of Padua, Padua, Italy
Bio-protocol author page: a4128
Rosario Rizzuto
Rosario RizzutoAffiliation: Department of Biomedical Sciences, University of Padua, Padua, Italy
For correspondence: rosario.rizzuto@unipd.it
Bio-protocol author page: a4129
 and Cristina Mammucari
Cristina MammucariAffiliation: Department of Biomedical Sciences, University of Padua, Padua, Italy
For correspondence: cristina.mammucari@unipd.it
Bio-protocol author page: a4130
date: 3/5/2017, 213 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2155.

Brief version appeared in EMBO Mol Med, May 2016
Aequorin is a Ca2+ sensitive photoprotein suitable to measure intracellular Ca2+ transients in mammalian cells. Thanks to recombinant cDNAs expression, aequorin can be specifically targeted to various subcellular compartments, thus allowing an accurate measurement of Ca2+ uptake and release of different intracellular organelles. Here, we describe how to use this probe to measure cytosolic Ca2+ levels and mitochondrial Ca2+ uptake in mammalian cells.

Laser Scanning Confocal Microcopy for Arabidopsis Epidermal, Mesophyll, and Vascular Parenchyma Cells

Featured protocol,  Authors: Christian Elowsky*
Christian ElowskyAffiliation: Department of Agronomy and Horticulture, University of Nebraska, Lincoln, USA
Bio-protocol author page: a4164
Yashitola Wamboldt*
Yashitola WamboldtAffiliation: Department of Agronomy and Horticulture, University of Nebraska, Lincoln, USA
Bio-protocol author page: a4165
 and Sally Mackenzie
Sally MackenzieAffiliation: Department of Agronomy and Horticulture, University of Nebraska, Lincoln, USA
For correspondence: smackenzie2@unl.edu
Bio-protocol author page: a4166
 (*contributed equally to this work) date: 3/5/2017, 288 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2150.

Brief version appeared in Mol Plant, Feb 2016
Investigation of protein targeting to plastids in plants by confocal laser scanning microscopy (CLSM) can be complicated by numerous sources of artifact, ranging from misinterpretations from in vivo protein over-expression, false fluorescence in cells under stress, and organellar mis-identification. Our studies have focused on the plant-specific gene MSH1, which encodes a dual targeting protein that is regulated in its expression and resides within the nucleoid of a specialized plastid type (Virdi et al., 2016). Therefore, our methods have been optimized to study protein dual targeting to mitochondria and plastids, spatial and temporal regulation of protein expression, and sub-organellar localization, producing a protocol and set of experimental standards that others may find useful for such studies.

Axonal Conduction Velocity Measurement

Featured protocol,  Authors: Margaret Louise DeMaegd
Margaret Louise DeMaegdAffiliation: School of Biological Sciences, Illinois State University, Normal, IL, USA
Bio-protocol author page: a4177
Carola Städele
Carola StädeleAffiliation: School of Biological Sciences, Illinois State University, Normal, IL, USA
For correspondence: carola@neurobiologie.de
Bio-protocol author page: a4150
 and Wolfgang Stein
Wolfgang SteinAffiliation: School of Biological Sciences, Illinois State University, Normal, IL, USA
For correspondence: wstein@neurobiologie.de
Bio-protocol author page: a4151
date: 3/5/2017, 180 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2152.

Brief version appeared in J Neurosci, Jun 2016
Action potential conduction velocity is the speed at which an action potential (AP) propagates along an axon. Measuring AP conduction velocity is instrumental in determining neuron health, function, and computational capability, as well as in determining short-term dynamics of neuronal communication and AP initiation (Ballo and Bucher, 2009; Bullock, 1951; Meeks and Mennerick, 2007; Rosenthal and Bezanilla, 2000; Städele and Stein, 2016; Swadlow and Waxman, 1976). Conduction velocity can be measured using extracellular recordings along the nerve through which the axon projects. Depending on the number of axons in the nerve, AP velocities of individual or many axons can be detected.

Isolation and Primary Culture of Adult Human Adipose-derived Stromal/Stem Cells

Featured protocol,  Authors: Robert B. Jones
Robert B. JonesAffiliation: Center for Stem Cell Research and Regenerative Medicine, Tulane University School of Medicine, New Orleans, USA
Bio-protocol author page: a4155
Amy L. Strong
Amy L. StrongAffiliation: Center for Stem Cell Research and Regenerative Medicine, Tulane University School of Medicine, New Orleans, USA
Bio-protocol author page: a4156
Jeffrey M. Gimble
Jeffrey M. GimbleAffiliation 1: Center for Stem Cell Research and Regenerative Medicine, Tulane University School of Medicine, New Orleans, USA
Affiliation 2: LaCell LLC, New Orleans, USA
Affiliation 3: Department of Surgery, Tulane University School of Medicine, New Orleans, USA
Bio-protocol author page: a4154
 and Bruce A. Bunnell
Bruce A. BunnellAffiliation 1: Center for Stem Cell Research and Regenerative Medicine, Tulane University School of Medicine, New Orleans, USA
Affiliation 2: Department of Pharmacology, Tulane University School of Medicine, New Orleans, USA
For correspondence: bbunnell@tulane.edu
Bio-protocol author page: a4157
date: 3/5/2017, 177 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2161.

Brief version appeared in Stem Cells, Mar 2016
Adipose-derived stromal/stem cells (ASCs) are multipotent cells that can be isolated from adipose tissue. Studies have shown that cells have the capacity to self-renew and differentiate into adipocyte, chondrocyte, myocyte, and osteoblast lineages. Thus, significant interest regarding their use for regenerative purposes to restore aging or damaged tissue has grown in recent decades. These cells have also been shown to immunomodulate the microenvironment and secrete abundant growth factors, which minimize inflammation and aid repair and regeneration. ASCs can be readily isolated from the stromal vascular fraction (SVF) of lipoaspirates. Given their ease of accessibility, bountiful source, and potential application in regenerative medicine and tissue engineering, there is growing interest in the characterization and utilization of ASCs. This protocol describes the isolation of ASCs from adult human adipose tissue as well as methods for culture maintenance including expansion and cryopreservation.

Liposome Flotation Assays for Phosphoinositide-protein Interaction

Featured protocol,  Authors: Helene Tronchere
Helene TronchereAffiliation: INSERM U1048 I2MC and Université Paul Sabatier, Toulouse, France
Bio-protocol author page: a4209
 and Frederic Boal
Frederic BoalAffiliation: INSERM U1048 I2MC and Université Paul Sabatier, Toulouse, France
For correspondence: frederic.boal@inserm.fr
Bio-protocol author page: a4210
date: 3/5/2017, 208 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2169.

Brief version appeared in J Cell Sci, Feb 2015
Phosphoinositides are rare membrane lipids involved in the control of the major cellular functions and signaling pathways. They are able to recruit specific effector proteins to the cytosolic face of plasma membrane and organelles to coordinate a vast variety of signaling and trafficking processes, as well to maintain specific identity of the different subcellular compartments (Di Paolo and De Camilli, 2006; Lemmon, 2003). Therefore, analysis of these effectors’ binding properties and specificity towards different phosphoinositides is crucial for the understanding of their cellular functions. This protocol describes a method to characterize the binding of proteins to different phosphoinositide-containing vesicles.

Surface Inoculation and Quantification of Pseudomonas syringae Population in the Arabidopsis Leaf Apoplast

Featured protocol,  Authors: Cristián Jacob*
Cristián JacobAffiliation: Department of Plant Sciences, University of California, Davis, USA
For correspondence: cjjacob@ucdavis.edu
Bio-protocol author page: a4204
Shweta Panchal*
Shweta PanchalAffiliation: Centre for Genome Research, Faculty of Science, the Maharaja Sayajirao University of Baroda, Baroda, India
For correspondence: shwetapanchal84@gmail.com
Bio-protocol author page: a4158
 and Maeli Melotto
Maeli MelottoAffiliation: Department of Plant Sciences, University of California, Davis, USA
For correspondence: melotto@ucdavis.edu
Bio-protocol author page: a4160
 (*contributed equally to this work) date: 3/5/2017, 239 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2167.

Brief version appeared in Front Plant Sci, Jun 2016
Bacterial pathogens must enter the plant tissue in order to cause a successful infection. Foliar bacterial pathogens that are not able to directly penetrate the plant epidermis rely on wounds or natural openings to internalize leaves. This protocol describes a procedure to estimate the population size of Pseudomonas syringae in the leaf apoplast after surface inoculation of Arabidopsis rosettes.

An HPLC-based Method to Quantify Coronatine Production by Bacteria

Featured protocol,  Authors: Shweta Panchal
Shweta PanchalAffiliation: Centre for Genome Research, Department of Microbiology and Biotechnology Centre, the Maharaja Sayajirao University of Baroda, Baroda, India
For correspondence: shwetapanchal84@gmail.com
Bio-protocol author page: a4158
Zachary S. Breitbach
Zachary S. BreitbachAffiliation: Department of Chemistry, University of Texas, Arlington, USA
For correspondence: zachbreitbach@yahoo.com
Bio-protocol author page: a4159
 and Maeli Melotto
Maeli MelottoAffiliation: Department of Plant Sciences, University of California, Davis, USA
For correspondence: melotto@ucdavis.edu
Bio-protocol author page: a4160
date: 3/5/2017, 232 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2147.

Brief version appeared in Front Plant Sci, Jun 2016
Coronatine is a polyketide phytotoxin produced by several pathovars of the plant pathogenic bacterium Pseudomonas syringae. It is one of the most important virulence factors determining the success of bacterial pathogenesis in the plant at both epiphytic and endophytic stages of the disease cycle. This protocol describes an optimized procedure to culture bacterial cells for coronatine production and to quantify the amount of coronatine secreted in the culture medium using an HPLC-based method.

Differential Salt Fractionation of Nuclei to Analyze Chromatin-associated Proteins from Cultured Mammalian Cells

Authors: Christin Herrmann
Christin HerrmannAffiliation 1: Division of Cancer Pathobiology, Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia, Philadelphia, USA
Affiliation 2: Cell & Molecular Biology Graduate Group, University of Pennsylvania, Philadelphia, USA
Bio-protocol author page: a4254
Daphne C. Avgousti
Daphne C. AvgoustiAffiliation 1: Division of Cancer Pathobiology, Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia, Philadelphia, USA
Affiliation 2: Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, USA
Bio-protocol author page: a4255
 and Matthew D. Weitzman
Matthew D. WeitzmanAffiliation 1: Division of Cancer Pathobiology, Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia, Philadelphia, USA
Affiliation 2: Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, USA
For correspondence: weitzmanm@email.chop.edu
Bio-protocol author page: a4256
date: 3/20/2017, 123 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2175.

[Abstract] Nucleosomes are the core units of cellular chromatin and are comprised of 147 base pairs (bp) of DNA wrapped around an octamer of histone proteins. Proteins such as chromatin remodelers, transcription factors, and DNA repair proteins interact dynamically with chromatin to regulate access to DNA, control gene transcription, and maintain genome integrity. ...

MHC Class II Tetramer Labeling of Human Primary CD4+ T Cells from HIV Infected Patients

Authors: Moran Galperin
Moran GalperinAffiliation: Pasteur Institute, Viral Pathogenesis Unit, Paris, France
For correspondence: moran.galperin@pasteur.fr
Bio-protocol author page: a4268
Daniela Benati
Daniela BenatiAffiliation: Center for Regenerative Medicine “Stephano Ferrari”, Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy
Bio-protocol author page: a4269
Mathieu Claireaux
Mathieu ClaireauxAffiliation: Pasteur Institute, Viral Pathogenesis Unit, Paris, France
Bio-protocol author page: a4270
Madhura Mukhopadhyay
Madhura MukhopadhyayAffiliation: Pasteur Institute, Viral Pathogenesis Unit, Paris, France
Bio-protocol author page: a4271
 and Lisa A. Chakrabarti
Lisa A. ChakrabartiAffiliation: Pasteur Institute, Viral Pathogenesis Unit, Paris, France
Bio-protocol author page: a4272
date: 3/20/2017, 119 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2187.

[Abstract] Major Histocompatibility Complex (MHC) tetramers have been used for two decades to detect, isolate and characterize T cells specific for various pathogens and tumor antigens. In the context of Human Immunodeficiency Virus (HIV) infection, antigen-specific CD8+ T cells have been extensively studied ex vivo, as they can be readily detected by HIV peptide-loaded ...

Adoptive Transfer of Lung Antigen Presenting Cells

Authors: Xiaofeng Zhou
Xiaofeng ZhouAffiliation: Department of Internal Medicine, Pulmonary and Critical Care Medicine Division, University of Michigan, Ann Arbor, Michigan, USA
For correspondence: xiazhou@umich.edu
Bio-protocol author page: a4212
 and Bethany B Moore
Bethany B MooreAffiliation: Department of Internal Medicine, Pulmonary and Critical Care Medicine Division, University of Michigan, Ann Arbor, Michigan, USA
Bio-protocol author page: a4213
date: 3/20/2017, 92 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2182.

[Abstract] Our protocol describes adoptive transfer of antigen presenting cells (APCs) isolated from the lungs by enzymatic digestion and magnetic enrichment. This protocol can be used to study APC functions and trafficking. ...

Mouse Model of Reversible Intestinal Inflammation

Authors: Cheong KC Kwong Chung
Cheong KC Kwong ChungAffiliation: Division of Experimental Pathology, Institute of Pathology, University of Bern, Bern, Switzerland
For correspondence: cheong.kwong@pathology.unibe.ch
Bio-protocol author page: a4194
Jennifer Brasseit
Jennifer BrasseitAffiliation: Division of Experimental Pathology, Institute of Pathology, University of Bern, Bern, Switzerland
Bio-protocol author page: a4190
Esther Althaus-Steiner
Esther Althaus-SteinerAffiliation: Division of Experimental Pathology, Institute of Pathology, University of Bern, Bern, Switzerland
Bio-protocol author page: a4191
Silvia Rihs
Silvia RihsAffiliation: Division of Experimental Pathology, Institute of Pathology, University of Bern, Bern, Switzerland
Bio-protocol author page: a4192
 and Christoph Mueller
Christoph MuellerAffiliation: Division of Experimental Pathology, Institute of Pathology, University of Bern, Bern, Switzerland
For correspondence: christoph.mueller@pathology.unibe.ch
Bio-protocol author page: a4193
date: 3/20/2017, 127 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2173.

[Abstract] Current therapies to treat inflammatory bowel disease by dampening excessive inflammatory immune responses have had limited success (Reinisch et al., 2011; Rutgeerts et al., 2005; Sandborn et al., 2012). To develop new therapeutic interventions, there is a need for better understanding of the mechanisms that are operative during mucosal healing (Pineton ...

Determination of the Predatory Capability of Bdellovibrio bacteriovorus HD100

Authors: Cristina Herencias
Cristina HerenciasAffiliation: Environmental Biology Department, Centro de Investigaciones Biológicas, CSIC, Madrid, Spain
Bio-protocol author page: a4258
M. Auxiliadora Prieto
M. Auxiliadora PrietoAffiliation: Environmental Biology Department, Centro de Investigaciones Biológicas, CSIC, Madrid, Spain
Bio-protocol author page: a4259
 and Virginia Martínez
Virginia MartínezAffiliation: Environmental Biology Department, Centro de Investigaciones Biológicas, CSIC, Madrid, Spain
Present address: Evolva Biotech A/S, Copenhagen, Denmark
For correspondence: virginiaml83@gmail.com
Bio-protocol author page: a4257
date: 3/20/2017, 116 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2177.

[Abstract] Bdellovibrio bacteriovorus HD100 is an obligate predator that preys upon a wide variety of Gram negative bacteria. The biphasic growth cycle of Bdellovibrio includes a free-swimming attack phase and an intraperiplasmic growth phase, where the predator replicates its DNA and grows using the prey as a source of nutrients, finally dividing into individual ...

In vitro Assay to Assess Efficacy of Potential Antiviral Compounds against Enterovirus D68

Authors: Liang Sun
Liang SunAffiliation: KU Leuven – University of Leuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, Leuven, Belgium
Bio-protocol author page: a4263
Leen Delang
Leen DelangAffiliation: KU Leuven – University of Leuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, Leuven, Belgium
Bio-protocol author page: a4264
Carmen Mirabelli
Carmen Mirabelli Affiliation: KU Leuven – University of Leuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, Leuven, Belgium
Bio-protocol author page: a4265
 and Johan Neyts
Johan NeytsAffiliation: KU Leuven – University of Leuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, Leuven, Belgium
For correspondence: johan.neyts@kuleuven.be
Bio-protocol author page: a1546
date: 3/20/2017, 85 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2183.

[Abstract] In 2014 enterovirus D68 (EV-D68) caused the largest outbreak in the United States since the discovery of the virus. Distinct from before, the 2014 infections were associated with more severe respiratory disease and occasional neurological complications. So far, there are no available vaccines or antivirals for the prophylaxis or treatment of EV-D68 ...

3D Stroma Invasion Assay

Authors: Yvette May Coulson-Thomas
Yvette May Coulson-ThomasAffiliation: Department of Biochemistry, Universidade Federal de São Paulo, São Paulo, Brazil
For correspondence: ycoulsonthomas@gmail.com
Bio-protocol author page: a4214
 and Vivien Jane Coulson-Thomas
Vivien Jane Coulson-ThomasAffiliation: College of Optometry, the Ocular Surface Institute (TOSI), University of Houston, Houston, USA
For correspondence: vcoulsonthomas@gmail.com
Bio-protocol author page: a1653
date: 3/20/2017, 99 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2195.

[Abstract] We have developed a 3D co-culture system composed of fibroblasts and colorectal cancer cells that enables us to study the desmoplastic reaction. This method also enables us to study the influence of the desmoplastic reaction on the migration of colorectal cancer cells through the surrounding stroma. This protocol has been previously published (Coulson-Thomas ...

Reprogram Murine Epiblast Stem Cells by Epigenetic Inhibitors

Authors: Hui Zhang
Hui ZhangAffiliation: Department of Pathology, University of Michigan, Ann Arbor, USA
Bio-protocol author page: a4176
 and Yali Dou
Yali DouAffiliation: Department of Pathology, University of Michigan, Ann Arbor, USA
For correspondence: yalid@med.umich.edu
Bio-protocol author page: a4171
date: 3/5/2017, 170 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2168.

[Abstract] Pluripotent stem cells in the naïve state are highly useful in regenerative medicine and tissue engineering. A robust reprogramming of the primed murine Epiblast Stem Cells (EpiSCs) to naïve pluripotency is feasible via chemical-only approach. This protocol described a method to reprogram murine EpiSCs by MM-401 treatment, which blocks histone H3K4 ...

Ca2+ Measurements in Mammalian Cells with Aequorin-based Probes

Authors: Anna Tosatto
Anna TosattoAffiliation: Department of Biomedical Sciences, University of Padua, Padua, Italy
Bio-protocol author page: a4128
Rosario Rizzuto
Rosario RizzutoAffiliation: Department of Biomedical Sciences, University of Padua, Padua, Italy
For correspondence: rosario.rizzuto@unipd.it
Bio-protocol author page: a4129
 and Cristina Mammucari
Cristina MammucariAffiliation: Department of Biomedical Sciences, University of Padua, Padua, Italy
For correspondence: cristina.mammucari@unipd.it
Bio-protocol author page: a4130
date: 3/5/2017, 213 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2155.

[Abstract] Aequorin is a Ca2+ sensitive photoprotein suitable to measure intracellular Ca2+ transients in mammalian cells. Thanks to recombinant cDNAs expression, aequorin can be specifically targeted to various subcellular compartments, thus allowing an accurate measurement of Ca2+ uptake and release of different intracellular organelles. Here, we describe how ...

Laser Scanning Confocal Microcopy for Arabidopsis Epidermal, Mesophyll, and Vascular Parenchyma Cells

Authors: Christian Elowsky*
Christian ElowskyAffiliation: Department of Agronomy and Horticulture, University of Nebraska, Lincoln, USA
Bio-protocol author page: a4164
Yashitola Wamboldt*
Yashitola WamboldtAffiliation: Department of Agronomy and Horticulture, University of Nebraska, Lincoln, USA
Bio-protocol author page: a4165
 and Sally Mackenzie
Sally MackenzieAffiliation: Department of Agronomy and Horticulture, University of Nebraska, Lincoln, USA
For correspondence: smackenzie2@unl.edu
Bio-protocol author page: a4166
 (*contributed equally to this work) date: 3/5/2017, 288 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2150.

[Abstract] Investigation of protein targeting to plastids in plants by confocal laser scanning microscopy (CLSM) can be complicated by numerous sources of artifact, ranging from misinterpretations from in vivo protein over-expression, false fluorescence in cells under stress, and organellar mis-identification. Our studies have focused on the plant-specific gene ...
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Scratch Wound Healing Assay

Author: Yanling Chen
Yanling ChenAffiliation: Department of Immunology, The Scripps Research Institute, La Jolla, USA
For correspondence: ylchen@scripps.edu
Bio-protocol author page: a27
date: 3/5/2012, 53941 views, 6 Q&A
DOI: https://doi.org/10.21769/BioProtoc.100.

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

Transwell Cell Migration Assay Using Human Breast Epithelial Cancer Cell

Author: Yanling Chen
Yanling ChenAffiliation: Department of Immunology, The Scripps Research Institute, La Jolla, USA
For correspondence: ylchen@scripps.edu
Bio-protocol author page: a27
date: 2/20/2012, 42738 views, 7 Q&A
DOI: https://doi.org/10.21769/BioProtoc.99.

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

In vitro Culture of Human PBMCs

Authors: Santosh K Panda
Santosh K PandaAffiliation: Infectious Disease Biology, Institute of Life Sciences, Bhubaneswar, India
Bio-protocol author page: a221
 and Balachandran Ravindran
Balachandran RavindranAffiliation: Infectious Disease Biology, Institute of Life Sciences, Bhubaneswar, India
For correspondence: ravindran8@gmail.com
Bio-protocol author page: a222
date: 2/5/2013, 40672 views, 4 Q&A
DOI: https://doi.org/10.21769/BioProtoc.322.

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

Detection of Intracellular Reactive Oxygen Species (CM-H2DCFDA)

Authors: Rabii Ameziane-El-Hassani
Rabii Ameziane-El-HassaniAffiliation 1: UBRM, Centre National de l'Energie, des Sciences et des Techniques Nucléaires, Rabat, Morocco
Affiliation 2: Institut Gustave Roussy, FRE2939 Centre National de la Recherche Scientifique, Villejuif, France
Bio-protocol author page: a200
 and Corinne Dupuy
Corinne DupuyAffiliation 1: Institut Gustave Roussy, FRE2939 Centre National de la Recherche Scientifique, Villejuif, France
Affiliation 2: University Paris, Sud 11, Orsay, France
For correspondence: dupuy@igr.fr
Bio-protocol author page: a201
date: 1/5/2013, 40612 views, 2 Q&A
DOI: https://doi.org/10.21769/BioProtoc.313.

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

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

Author: Hui Zhu
Hui ZhuAffiliation: Department of Genetics, Stanford University, Stanford, USA
For correspondence: huizhu@stanford.edu
Bio-protocol author page: a32
date: 4/5/2012, 38499 views, 2 Q&A
DOI: https://doi.org/10.21769/BioProtoc.198.

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

Clonogenic Assay

Author: Xiaodong Yang
Xiaodong YangAffiliation: Department of Neurology, University of California, San Francisco, USA
For correspondence: yangxiaodong1@yahoo.com
Bio-protocol author page: a43
date: 5/20/2012, 36971 views, 5 Q&A
DOI: https://doi.org/10.21769/BioProtoc.187.

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

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

Author: Ran Chen
Ran ChenAffiliation: Department of Genetics, Stanford University, Stanford, USA
For correspondence: rcchen@jfkbio.com
Bio-protocol author page: a34
date: 2/5/2012, 34849 views, 15 Q&A
DOI: https://doi.org/10.21769/BioProtoc.68.

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

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

Authors: Josephine MY Ko
Josephine MY KoAffiliation: Clinical Oncology Department, The University of Hong Kong, Hong Kong , Hong Kong SAR
Bio-protocol author page: a100
 and Maria Li Lung
Maria Li LungAffiliation: Clinical Oncology Department, The Univerisity of Hong Kong, Hong Kong, Hong Kong SAR
For correspondence: mlilung@hku.hk
Bio-protocol author page: a101
date: 9/20/2012, 31960 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.260.

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

Soft–Agar colony Formation Assay

Author: FengZhi Liu
FengZhi LiuAffiliation: School of Biomedical Sciences, Thomas Jefferson University, Philadelphia, USA
For correspondence: fengzhi6@yahoo.com
Bio-protocol author page: a51
date: 7/5/2012, 30860 views, 6 Q&A
DOI: https://doi.org/10.21769/BioProtoc.220.

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

[Bio101] Cell Adhesion Assay

Author: Yanling Chen
Yanling ChenAffiliation: Department of Immunology, The Scripps Research Institute, La Jolla, USA
For correspondence: ylchen@scripps.edu
Bio-protocol author page: a27
date: 3/5/2012, 27733 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.98.

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