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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.

Mouse CD8+ T Cell Migration in vitro and CXCR3 Internalization Assays

Featured protocol,  Authors: Rosa Barreira da Silva
Rosa Barreira da SilvaAffiliation: Cancer Immunology, Genentech, South San Francisco, USA
For correspondence: albertm7@gene.com
Bio-protocol author page: a4161
 and Matthew L. Albert
Matthew L. AlbertAffiliation 1: Cancer Immunology, Genentech, South San Francisco, USA
Affiliation 2: Laboratory of Dendritic Cell Immunobiology, Institut Pasteur, Paris, France
Affiliation 3: INSERM U1223, Paris, France
Bio-protocol author page: a4163
date: 3/20/2017, 100 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2185.

Brief version appeared in Nat Immunol, Aug 2015
Chemokines are molecules that regulate the positioning of cells during homeostasis and inflammation. CXCL10 is an interferon-induced chemokine that attracts cells that express the chemokine receptor CXCR3 on their surface. CXCL10 expression is often induced upon inflammation and guides lymphocytes, such as T and NK cells, into the injured tissues. Notably, CXCL10 binding to CXCR3 induces receptor internalization and, therefore, low CXCR3 levels in cells positive for CXCR3 expression can be indicative of chemokine signaling.

Measurement of Dipeptidylpeptidase Activity in vitro and in vivo

Featured protocol,  Authors: Rosa Barreira da Silva
Rosa Barreira da SilvaAffiliation: Cancer Immunology, Genentech, South San Francisco, CA, USA
Bio-protocol author page: a4161
Molly A. Ingersoll
Molly A. IngersollAffiliation 1: Laboratory of Dendritic Cell Immunobiology, Institut Pasteur, Paris, France
Affiliation 2: INSERM U1223, Paris, France
Bio-protocol author page: a4162
 and Matthew L. Albert
Matthew L. AlbertAffiliation 1: Cancer Immunology, Genentech, South San Francisco, CA, USA
Affiliation 2: Laboratory of Dendritic Cell Immunobiology, Institut Pasteur, Paris, France
Affiliation 3: INSERM U1223, Paris, France
For correspondence: albertm7@gene.com
Bio-protocol author page: a4163
date: 3/20/2017, 110 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2184.

Brief version appeared in Nat Immunol, Aug 2015
Dipeptidylpeptidases (DPPs) are serine proteases, which cleave small proteins and peptides possessing a proline or an alanine in the second position of their N-terminus. Among the members of this family, dipeptidylpeptidase 4 (DPP4) is constitutively expressed in the extracellular space. DPP4 is found at the surface of many hematopoietic and non-hematopoietic cells and is also present in many biological fluids in a bioactive soluble form. DPP4 expression is modulated by inflammation, and measurements of its activity have been used as biomarker for disease. Here, we describe a method to evaluate the enzymatic activity of DPP4 in vitro and in vivo.

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.

RNA-protein UV-crosslinking Assay

Featured protocol,  Authors: Dipak Kumar Poria
Dipak Kumar PoriaAffiliation: Department of Biological Sciences, Indian Institute of Science Education and Research, Kolkata, India
Bio-protocol author page: a4215
 and Partho Sarothi Ray
Partho Sarothi RayAffiliation: Department of Biological Sciences, Indian Institute of Science Education and Research, Kolkata, India
For correspondence: psray@iiserkol.ac.in
Bio-protocol author page: a4216
date: 3/20/2017, 173 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2193.

Brief version appeared in Oncogene, Mar 2016
RNA-protein interactions play a crucial role in every aspect of RNA metabolism, and also plays a major role in post-transcriptional gene regulation. RNA-binding proteins have been implicated in viral gene expression (Ray and Das, 2002) and microRNA-mediated gene regulation (Poria et al., 2016). Here we have described the protocol which (1) covalently links transiently interacting RNA-protein complexes by UV crosslinking, (2) removes the unprotected RNA by RNase digestion and (3) detects the RNA-protein complexes by SDS-PAGE analysis. This protocol provides a rapid and reliable means to directly assay RNA-protein interactions and their kinetics using purified proteins and also help in identifying novel RNA-protein interactions

Polysome Analysis

Featured protocol,  Authors: Dipak Kumar Poria
Dipak Kumar PoriaAffiliation: Department of Biological Sciences, Indian Institute of Science Education and Research, Kolkata, India
Bio-protocol author page: a4215
 and Partho Sarothi Ray
Partho Sarothi RayAffiliation: Department of Biological Sciences, Indian Institute of Science Education and Research, Kolkata, India
For correspondence: psray@iiserkol.ac.in
Bio-protocol author page: a4216
date: 3/20/2017, 144 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2192.

Brief version appeared in Oncogene, Mar 2016
Polysome analysis is a method to separate mRNAs from a cell into actively translating and non-translating fractions depending on their association with polysomes. By this protocol, cell lysates are fractionated by sucrose density gradient ultracentrifugation. Free mRNA fraction and various ribosomal fractions, such as 40S, 60S, monosomes and polysomes are collected by fractionation. Association of particular mRNAs with these fractions is detected by reverse transcription – PCR to investigate the translational state of the mRNA.

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).

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.

Thinned-skulled Cranial Window Preparation (Mice)

Featured protocol,  Authors: Lifeng Zhang
Lifeng ZhangAffiliation: Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Baltimore, USA
For correspondence: lfzhang916@hotmail.com
Bio-protocol author page: a4186
Bo Liang
Bo LiangAffiliation: Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Baltimore, USA
Bio-protocol author page: a4187
Yun Li
Yun LiAffiliation: Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Baltimore, USA
Bio-protocol author page: a4188
 and Da-Ting Lin
Da-Ting LinAffiliation 1: Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Baltimore, USA
Affiliation 2: The Jackson Laboratory, Bar Harbor, USA
Affiliation 3: The Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, USA
Bio-protocol author page: a4189
date: 3/5/2017, 159 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2158.

Brief version appeared in Nat Neurosci, Apr 2016
Imaging structural plasticity or activity of neurons in the brain circuit will facilitate understanding the neural mechanisms underlying animal behavior. Here we describe a modified procedure, the polished and reinforced thinned-skull cranial window preparation, by which we can image dendrites and spines in mouse layer I cortex for weeks (Zhang et al., 2016). By this method, we also imaged the glioma initiation in the mouse cortex for two weeks in previous work (Zhang et al., 2012), which included the photographs and video for reference.

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.

Olfactory Habituation-dishabituation Test (Mouse)

Featured protocol,  Authors: Hiroo Takahashi
Hiroo TakahashiAffiliation: Laboratory for Molecular Biology of Neural System, Advanced Medical Research Center, Nara Medical University , Kashihara, Nara, Japan
Bio-protocol author page: a4174
 and Akio Tsuboi
Akio TsuboiAffiliation: Laboratory for Molecular Biology of Neural System, Advanced Medical Research Center, Nara Medical University , Kashihara, Nara, Japan
For correspondence: atsuboi@naramed-u.ac.jp
Bio-protocol author page: a4175
date: 3/5/2017, 162 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2154.

Brief version appeared in J Neurosci, Aug 2016
Olfaction plays a fundamental role for the various behaviors such as feeding, mating, nursing, and avoidance in mice. Behavioral tests that characterize abilities of odor detection and recognition using genetically modified mice reveal the contribution of target genes to the olfactory processing. Here, we describe the olfactory habituation-dishabituation test for investigating the odor detection threshold in mice.

Olfactory Avoidance Test (Mouse)

Featured protocol,  Authors: Hiroo Takahashi
Hiroo TakahashiAffiliation: Laboratory for Molecular Biology of Neural System, Advanced Medical Research Center, Nara Medical University , Kashihara, Nara, Japan
Bio-protocol author page: a4174
 and Akio Tsuboi
Akio TsuboiAffiliation: Laboratory for Molecular Biology of Neural System, Advanced Medical Research Center, Nara Medical University , Kashihara, Nara, Japan
For correspondence: atsuboi@naramed-u.ac.jp
Bio-protocol author page: a4175
date: 3/5/2017, 150 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2153.

Brief version appeared in J Neurosci, Aug 2016
In mice, olfaction plays a pivotal role for the various behaviors, such as feeding, mating, nursing and avoidance. Behavioral tests that analyze abilities of odor detection and recognition using genetically modified mice reveal the contribution of target genes to the olfactory processing. Here, we describe the olfactory avoidance test to investigate the odor detection ability in mice.

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.

Extracellular Axon Stimulation

Featured protocol,  Authors: Carola Städele
Carola StädeleAffiliation: School of Biological Sciences, Illinois State University, Normal, IL, USA
For correspondence: carola@neurobiologie.de
Bio-protocol author page: a4150
Margaret Louise DeMaegd
Margaret Louise DeMaegdAffiliation: School of Biological Sciences, Illinois State University, Normal, IL, USA
Bio-protocol author page: a4177
 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, 177 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2151.

Brief version appeared in J Neurosci, Jun 2016
This is a detailed protocol explaining how to perform extracellular axon stimulations as described in Städele and Stein, 2016. The ability to stimulate and record action potentials is essential to electrophysiological examinations of neuronal function. Extracellular stimulation of axons traveling in fiber bundles (nerves) is a classical technique in brain research and a fundamental tool in neurophysiology (Abbas and Miller, 2004; Barry, 2015; Basser and Roth, 2000; Cogan, 2008). It allows for activating action potentials in individual or multiple axons, controlling their firing frequency, provides information about the speed of neuronal communication, and neuron health and function.

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.

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 ...

Mouse CD8+ T Cell Migration in vitro and CXCR3 Internalization Assays

Authors: Rosa Barreira da Silva
Rosa Barreira da SilvaAffiliation: Cancer Immunology, Genentech, South San Francisco, USA
For correspondence: albertm7@gene.com
Bio-protocol author page: a4161
 and Matthew L. Albert
Matthew L. AlbertAffiliation 1: Cancer Immunology, Genentech, South San Francisco, USA
Affiliation 2: Laboratory of Dendritic Cell Immunobiology, Institut Pasteur, Paris, France
Affiliation 3: INSERM U1223, Paris, France
Bio-protocol author page: a4163
date: 3/20/2017, 100 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2185.

[Abstract] Chemokines are molecules that regulate the positioning of cells during homeostasis and inflammation. CXCL10 is an interferon-induced chemokine that attracts cells that express the chemokine receptor CXCR3 on their surface. CXCL10 expression is often induced upon inflammation and guides lymphocytes, ...

Measurement of Dipeptidylpeptidase Activity in vitro and in vivo

Authors: Rosa Barreira da Silva
Rosa Barreira da SilvaAffiliation: Cancer Immunology, Genentech, South San Francisco, CA, USA
Bio-protocol author page: a4161
Molly A. Ingersoll
Molly A. IngersollAffiliation 1: Laboratory of Dendritic Cell Immunobiology, Institut Pasteur, Paris, France
Affiliation 2: INSERM U1223, Paris, France
Bio-protocol author page: a4162
 and Matthew L. Albert
Matthew L. AlbertAffiliation 1: Cancer Immunology, Genentech, South San Francisco, CA, USA
Affiliation 2: Laboratory of Dendritic Cell Immunobiology, Institut Pasteur, Paris, France
Affiliation 3: INSERM U1223, Paris, France
For correspondence: albertm7@gene.com
Bio-protocol author page: a4163
date: 3/20/2017, 110 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2184.

[Abstract] Dipeptidylpeptidases (DPPs) are serine proteases, which cleave small proteins and peptides possessing a proline or an alanine in the second position of their N-terminus. Among the members of this family, dipeptidylpeptidase 4 (DPP4) is constitutively expressed in the extracellular space. DPP4 is found ...

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 ...

RNA-protein UV-crosslinking Assay

Authors: Dipak Kumar Poria
Dipak Kumar PoriaAffiliation: Department of Biological Sciences, Indian Institute of Science Education and Research, Kolkata, India
Bio-protocol author page: a4215
 and Partho Sarothi Ray
Partho Sarothi RayAffiliation: Department of Biological Sciences, Indian Institute of Science Education and Research, Kolkata, India
For correspondence: psray@iiserkol.ac.in
Bio-protocol author page: a4216
date: 3/20/2017, 173 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2193.

[Abstract] RNA-protein interactions play a crucial role in every aspect of RNA metabolism, and also plays a major role in post-transcriptional gene regulation. RNA-binding proteins have been implicated in viral gene expression (Ray and Das, 2002) and microRNA-mediated gene regulation (Poria et al., 2016). Here ...

Polysome Analysis

Authors: Dipak Kumar Poria
Dipak Kumar PoriaAffiliation: Department of Biological Sciences, Indian Institute of Science Education and Research, Kolkata, India
Bio-protocol author page: a4215
 and Partho Sarothi Ray
Partho Sarothi RayAffiliation: Department of Biological Sciences, Indian Institute of Science Education and Research, Kolkata, India
For correspondence: psray@iiserkol.ac.in
Bio-protocol author page: a4216
date: 3/20/2017, 144 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2192.

[Abstract] Polysome analysis is a method to separate mRNAs from a cell into actively translating and non-translating fractions depending on their association with polysomes. By this protocol, cell lysates are fractionated by sucrose density gradient ultracentrifugation. Free mRNA fraction and various ribosomal ...

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 ...

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. ...

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 ...
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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] 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, 30329 views, 18 Q&A
DOI: https://doi.org/10.21769/BioProtoc.157.

[Abstract] In vitro differentiation of helper T cells of various lineages is frequently used in T helper cell study. Naïve CD4 T cells can differentiate into certain lineage of T help cells in vitro in the presence of specific stimulatory cytokines and inhibition of cytokines that are essential for the differentiation ...
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