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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.

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).

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. ...

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, such as T and NK cells, into the injured tissues. ...

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 at the surface of many hematopoietic and non-hematopoietic ...

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 ...

Protocol for Murine/Mouse Platelets Isolation and Their Reintroduction in vivo

Authors: Jae Hong Im
Jae Hong ImAffiliation: CRUK-MRC Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK
Bio-protocol author page: a4021
 and Ruth J. Muschel
Ruth J. MuschelAffiliation: CRUK-MRC Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK
For correspondence: ruth.muschel@oncology.ox.ac.uk
Bio-protocol author page: a4022
date: 2/20/2017, 345 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2132.

[Abstract] Platelets and coagulation have long been known to be essential for metastasis in experimental models. In order to study the interactions between tumor cells, platelets and endothelium, we have adapted methods used in coagulation research for the isolation of platelets and their reintroduction into mice. Anti-coagulated murine blood served as the source ...

Analysis of the Virulence of Uropathogenic Escherichia coli Strain CFT073 in the Murine Urinary Tract

Authors: Anna Waldhuber
Anna WaldhuberAffiliation: Molekulare Pädiatrie, Dr. von Haunersches Kinderspital, Ludwig-Maximilians-Universität, München, Germany
Bio-protocol author page: a4062
Manoj Puthia
Manoj PuthiaAffiliation: Department of Microbiology, Immunology and Glycobiology (MIG), Institute of Laboratory Medicine, Lund University, Lund, Sweden
Bio-protocol author page: a4063
Andreas Wieser
Andreas WieserAffiliation: Max von Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie, Ludwig-Maximilians-Universität, München, Germany
Bio-protocol author page: a4064
Catharina Svanborg
Catharina SvanborgAffiliation: Department of Microbiology, Immunology and Glycobiology (MIG), Institute of Laboratory Medicine, Lund University, Lund, Sweden
Bio-protocol author page: a4065
 and Thomas Miethke
Thomas Miethke Affiliation: Institute of Medical Microbiology and Hygiene, Medical Faculty of Mannheim, University of Heidelberg, Mannheim, Germany
For correspondence: thomas.miethke@medma.uni-heidelberg.de
Bio-protocol author page: a4056
date: 2/5/2017, 379 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2129.

[Abstract] This urinary tract infection model was used to monitor the efficacy of a new virulence factor of the uropathogenic Escherichia coli strain CFT073 in vivo. The new virulence factor which we designated TIR-containing protein C (TcpC) blocks Toll-like receptor signaling and the NLRP3 inflammasome signaling cascade by interacting with key components of ...

Virus Binding and Internalization Assay for Adeno-associated Virus

Authors: Garrett E. Berry
Garrett E. BerryAffiliation 1: Gene Therapy Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
Affiliation 2: Curriculum in Genetics and Molecular Biology, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
For correspondence: berrygar@email.unc.edu
Bio-protocol author page: a3988
 and Longping V. Tse
Longping V. TseAffiliation: Gene Therapy Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
Bio-protocol author page: a940
date: 1/20/2017, 432 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2110.

[Abstract] The binding and internalization of adeno-associated virus (AAV) is an important determinant of viral infectivity and tropism. The ability to dissect these two tightly connected cellular processes would allow better understanding and provide insight on virus entry and trafficking. In the following protocol, we describe a quantitative PCR (qPCR) based ...

Isolation of PBMCs Using Vacutainer® Cellular Preparation Tubes (CPTTM)

Authors: Alaina Puleo
Alaina PuleoAffiliation: Institute for Immunity, Transplantation, and Infection, Stanford University School of Medicine, Stanford, Palo Alto, CA, USA
Bio-protocol author page: a4007
Chantia Carroll
Chantia CarrollAffiliation: Institute for Immunity, Transplantation, and Infection, Stanford University School of Medicine, Stanford, Palo Alto, CA, USA
Bio-protocol author page: a4008
Holden Maecker
Holden MaeckerAffiliation: Institute for Immunity, Transplantation, and Infection, Stanford University School of Medicine, Stanford, Palo Alto, CA, USA
For correspondence: maecker@stanford.edu
Bio-protocol author page: a1861
 and Rohit Gupta
Rohit GuptaAffiliation: Institute for Immunity, Transplantation, and Infection, Stanford University School of Medicine, Stanford, Palo Alto, CA, USA
Bio-protocol author page: a1867
date: 1/20/2017, 621 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2103.

[Abstract] Peripheral blood mononuclear cell (PBMC) isolation is commonly done via density gradient centrifugation over Ficoll-Hypaque, a labor-intensive procedure that requires skilled technicians and can contribute to sample variability. Cellular Preparation Tubes (CPTs) are Vacutainer blood draw tubes that contain Ficoll-Hypaque and a gel plug that separates ...
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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 ...

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 ...

[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 ...

[Bio101] Whole Blood Staining of Human Monocyte Subsets for Flow Cytometry

Author: Zheng Liu
Zheng LiuAffiliation: The Feinstein Institute for Medical Research, Manhasset, NY, USA
For correspondence: zl2119@caa.columbia.edu
Bio-protocol author page: a12
Anne Davidson Lab, date: 5/20/2011, 21612 views, 10 Q&A
DOI: https://doi.org/10.21769/BioProtoc.69.

[Abstract] This is a general protocol to stain whole human blood for flow analysis with minimal spontaneous activation of monocytes. This protocol was developed or modified in Dr. Anne Davidson’s lab at Feinstein Institute for Medical Research....

Isolation 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, 19811 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.323.

[Abstract] Peripheral blood mononuclear cells (PBMCs) are chiefly lymphocytes and monocytes. PBMCs are separated from the whole blood by a density gradient centrifugation method using Ficoll Histopaque....

[Bio101] In vivo BrdU Incorporation and Detection in Mouse

Author: Zheng Liu
Zheng LiuAffiliation: The Feinstein Institute for Medical Research, Manhasset, NY, USA
For correspondence: zl2119@caa.columbia.edu
Bio-protocol author page: a12
Anne Davidson Lab, date: 6/5/2011, 19455 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.81.

[Abstract] BrdU (Bromodeoxyuridine or 5-bromo-2’-deoxyuridine) is a synthetic nucleoside that is incorporated into DNA by proliferating cells. This protocol is to be used to incorporate and detect BrdU in murine plasma cells. The plasma cells described in this protocol are formed spontaneously in autoimmune mice ...

[Bio101] Thioglycollate Induced Peritonitis

Author: Zheng Liu
Zheng LiuAffiliation: The Feinstein Institute for Medical Research, Manhasset, NY, USA
For correspondence: zl2119@caa.columbia.edu
Bio-protocol author page: a12
Anne Davidson Lab, date: 6/20/2011, 17410 views, 3 Q&A
DOI: https://doi.org/10.21769/BioProtoc.84.

[Abstract] Intraperitoneal (i.p.) injection of thioglycollate elicits a robust influx of neutrophils into peritoneal cavity. The trafficking of the cells is believed to be mediated by chemokines CXCL1, CXCL2, and CXCL8 (Call et al., 2001; Cacalano et al., 1994). Thus this model can be used to test the ability ...

Intracellular Cytokine (INF-gamma) Staining Assay

Author: Huagang Zhang
Huagang ZhangAffiliation: Albert Einstein College of Medicine, Yeshiva University, New York City, USA
For correspondence: huagangzhang@gmail.com
Bio-protocol author page: a21
date: 4/5/2012, 17140 views, 2 Q&A
DOI: https://doi.org/10.21769/BioProtoc.122.

[Abstract] An intracellular cytokine (INF-gamma) staining assay is used to analyze the function of lymphocytes at the single cell level. By combining surface staining and intracellular cytokine staining, this assay can reveal the percentage of cytokine-releasing cells in a particular population, which cannot be ...

Isolation of Dendritic Cells and Macrophages from the Murine Kidneys of Lupus by Cell Sorter

Authors: Ramalingam Bethunaickan
Ramalingam BethunaickanAffiliation: Center for Autoimmunity and Musculoskeletal Diseases, Feinstein Institute for Medical Research, New York, USA
For correspondence: bramalingam@gmail.com
Bio-protocol author page: a24
 and Anne Davidson
Anne DavidsonAffiliation: Center for Autoimmunity and Musculoskeletal Diseases, Feinstein Institute for Medical Research, New York, USA
Bio-protocol author page: a1712
Anne Davidson Lab, date: 4/20/2012, 12715 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.168.

[Abstract] Methods for the isolation and characterization of mononuclear phagocytes from the kidneys of mice with SLE are essential to understand the patho-physiology of the disease. Activation of these cells is associated with the onset of clinical disease in mice and infiltration with these cells is associated ...

Bronchoalveolar Lavage and Lung Tissue Digestion

Authors: Hongwei Han
Hongwei HanAffiliation: Immunology Program, Benaroya Research Institute, Seattle, USA
For correspondence: hhan@benaroyaresearch.org
Bio-protocol author page: a544
 and Steven F. Ziegler
Steven F. ZieglerAffiliation: Immunology Program, Benaroya Research Institute, Seattle, USA
For correspondence: sziegler@benaroyaresearch.org
Bio-protocol author page: a543
date: 8/20/2013, 12228 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.859.

[Abstract] Bronchoalveolar lavage (BAL) is a simple but valuable and typically performed technique commonly used for studying the pathogenesis of lung diseases such as asthma and COPD. Cell counts can be combined with new methods for examining inflammatory responses, such as ELISA, Flow cytometric analysis, immunohistochemistry, ...
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