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

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

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

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

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

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

Analysis of Phagosomal Antigen Degradation by Flow Organellocytometry

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

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

Determination of H2O2 Generation by pHPA Assay

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

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

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

Featured protocol,  Authors: Jennifer L. Larson-Casey
Jennifer L. Larson-CaseyAffiliation: Department of Medicine, Division of Pulmonary, Allergy, and Critical Care Medicine, University of Alabama at Birmingham, Birmingham, AL, USA
Bio-protocol author page: a3716
 and A. Brent Carter
A. Brent CarterAffiliation 1: Department of Medicine, Division of Pulmonary, Allergy, and Critical Care Medicine, University of Alabama at Birmingham, Birmingham, AL, USA
Affiliation 2: Birmingham Veterans Administration Medical Center, Birmingham, AL, USA
For correspondence: bcarter1@uab.edu
Bio-protocol author page: a3717
date: 11/20/2016, 152 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2009.

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

Establishment of Patient-Derived Xenografts in Mice

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

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

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

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

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

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

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

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

[Background] This protocol was developed in order to assess the changes of ...

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

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

[Abstract] Antigen presentation by MHC class I molecules, also referred to as cross-presentation, elicits cytotoxic immune responses. In particular, dendritic cells (DC) are the most proficient cross-presenting cells, since they have developed unique means to control phagocytic and degradative pathways.
   This protocol allows the evaluation of antigen cross-presentation ...

Analysis of Phagosomal Antigen Degradation by Flow Organellocytometry

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

[Abstract] Professional phagocytes internalize self and non-self particles by phagocytosis to initiate innate immune responses. After internalization, the formed phagosome matures through fusion and fission events with endosomes and lysosomes to obtain a more acidic, oxidative and hydrolytic environment for the degradation of its cargo. Interestingly, phagosome ...

Determination of H2O2 Generation by pHPA Assay

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

[Abstract] The production of reactive oxygen species, including H2O2, is a process that can be used in signaling, cell death, or immune response. To quantify oxidative stress in cells, a fluorescence technique has been modified from a previously described method to measure H2O2 release from cells (Panus et al., 1993; Murthy et al., 2010; Larson-Casey et al., ...

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

Authors: Jennifer L. Larson-Casey
Jennifer L. Larson-CaseyAffiliation: Department of Medicine, Division of Pulmonary, Allergy, and Critical Care Medicine, University of Alabama at Birmingham, Birmingham, AL, USA
Bio-protocol author page: a3716
 and A. Brent Carter
A. Brent CarterAffiliation 1: Department of Medicine, Division of Pulmonary, Allergy, and Critical Care Medicine, University of Alabama at Birmingham, Birmingham, AL, USA
Affiliation 2: Birmingham Veterans Administration Medical Center, Birmingham, AL, USA
For correspondence: bcarter1@uab.edu
Bio-protocol author page: a3717
date: 11/20/2016, 152 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2009.

[Abstract] Because transforming growth factor-β (TGF-β1) induces differentiation of fibroblasts to myofibroblasts, we developed a protocol to evaluate alveolar macrophage-derived TGF-β1 regulation of lung fibroblast differentiation (Larson-Casey et al., 2016). The protocol evaluates the ability of mouse bronchoalveolar lavage (BAL) fluid to alter fibroblast differentiation. ...

Establishment of Patient-Derived Xenografts in Mice

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

[Abstract] Patient-derived xenograft (PDX) models for cancer research have recently attracted considerable attention in both the academy and industry (Hidalgo et al., 2014; Wilding and Bodmer, 2014). PDX models have been developed from different tumor types including lung cancer to improve the drug development process. These models are used for pre-clinical drug ...

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

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

[Abstract] In this protocol, we describe the method for isolating highly pure primary alveolar epithelial type II (ATII) cells from lungs of naïve mice. The method combines negative selection for a variety of lineage markers along with positive selection for EpCAM, a pan-epithelial cell marker. This method yields 2-3 x 106 ATII cells per mouse lung. The cell ...

Reconstruction of the Mouse Inflammasome System in HEK293T Cells

Authors: Hexin Shi
Hexin ShiAffiliation: Center for the Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, Texas, USA
Bio-protocol author page: a3655
Anne Murray
Anne Murray Affiliation: Center for the Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, Texas, USA
Bio-protocol author page: a3656
 and Bruce Beutler
Bruce BeutlerAffiliation: Center for the Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, Texas, USA
For correspondence: Bruce.Beutler@utsouthwestern.edu
Bio-protocol author page: a3657
date: 11/5/2016, 175 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.1986.

[Abstract] The NLRP3 (NLR family, Pyrin domain containing 3) inflammasome is a multiprotein complex comprised of NLRP3, pro-caspase-1, the adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC), and the protein kinase NIMA related kinase 7 (NEK7) (Shi et al., 2016; He et al., 2016; Schmid-Burgk et al., 2016). When cells are exposed to ...

Phagocytosis Assay to Measure Uptake of Necroptotic Cancer Cells by BMDCs

Authors: Tania Løve Aaes
Tania Løve AaesAffiliation 1: Molecular Signaling and Cell Death Unit, VIB Inflammation Research Center, Ghent, Belgium
Affiliation 2: Department of Biomedical Molecular Biology, Cancer Research Institute Ghent (CRIG), Ghent University, Ghent, Belgium
Bio-protocol author page: a3682
Dmitri V. Krysko
Dmitri V. KryskoAffiliation 1: Molecular Signaling and Cell Death Unit, VIB Inflammation Research Center, Ghent, Belgium
Affiliation 2: Department of Biomedical Molecular Biology, Cancer Research Institute Ghent (CRIG), Ghent University, Ghent, Belgium
Affiliation 3: Department of Basic Medical Sciences, Faculty of Medicine and Health Sciences, Ghent University, Ghent, Belgium
Bio-protocol author page: a3683
 and Peter Vandenabeele
Peter VandenabeeleAffiliation 1: Molecular Signaling and Cell Death Unit, VIB Inflammation Research Center, Ghent, Belgium
Affiliation 2: Department of Biomedical Molecular Biology, Cancer Research Institute Ghent (CRIG), Ghent University, Ghent, Belgium
Affiliation 3: Methusalem Program, Ghent University, Ghent, Belgium
For correspondence: peter.vandenabeele@irc.vib-ugent.be
Bio-protocol author page: a3684
date: 11/5/2016, 190 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.1997.

[Abstract] This protocol is a flow cytometry-based method to measure the phagocytosis efficiency of necroptotic target cells by bone marrow-derived dendritic cells (BMDCs) in vitro (Aaes et al., 2016). The method is a slightly modified and updated version of the protocols used in previously published papers (Krysko et al., 2006; Brouckaert et al., 2004). In brief, ...

Quantification of Tumor Material Uptake

Authors: Richard N. Hanna
Richard N. HannaAffiliation 1: Department of Respiratory, Inflammation and Autoimmune Diseases, MedImmune, LLC, Gaithersburg, USA
Affiliation 2: Division of Inflammation Biology, La Jolla Institute for Allergy and Immunology, La Jolla, USA
For correspondence: hannar@Medimmune.com
Bio-protocol author page: a3623
 and Catherine C. Hedrick
Catherine C. HedrickAffiliation: Division of Inflammation Biology, La Jolla Institute for Allergy and Immunology, La Jolla, USA
Bio-protocol author page: a3625
date: 10/20/2016, 284 views, 1 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.1974.

[Abstract] Extracellular tumor material including exosomes, microvesicles and apoptotic tumor debris may help cancers invade new organs. Enhancing the removal of extracellular tumor material by immune cells represents a novel immunotherapy approach for preventing cancer metastasis. This protocol quantifies the uptake and removal of extracellular tumor material ...
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 

In vitro Culture of Human PBMCs

Authors: Santosh K Panda
Santosh K PandaAffiliation: Infectious Disease Biology, Institute of Life Sciences, Bhubaneswar, India
Bio-protocol author page: a221
 and Balachandran Ravindran
Balachandran RavindranAffiliation: Infectious Disease Biology, Institute of Life Sciences, Bhubaneswar, India
For correspondence: ravindran8@gmail.com
Bio-protocol author page: a222
date: 2/5/2013, 35698 views, 4 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.322.

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

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

Author: Ran Chen
Ran ChenAffiliation: Department of Genetics, Stanford University, Stanford, USA
For correspondence: rcchen@jfkbio.com
Bio-protocol author page: a34
date: 2/5/2012, 33592 views, 15 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.68.

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

[Bio101] In vitro Differentiation of Mouse Th0, Th1 and Th2 from Naïve CD4 T Cells

Author: Jia Li
Jia LiAffiliation: Department of Immunology, Medical Center, Duke University, Durham, North Carolina, USA
For correspondence: jiali.email@gmail.com
Bio-protocol author page: a16
date: 11/20/2011, 28746 views, 18 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.157.

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

[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, 20478 views, 10 Q&A, How to cite
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....

[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, 18256 views, 0 Q&A, How to cite
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 ...

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, 17172 views, 0 Q&A, How to cite
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] 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, 16319 views, 3 Q&A, How to cite
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, 16260 views, 2 Q&A, How to cite
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, 11996 views, 0 Q&A, How to cite
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 ...

Isolation, Purification, and Culture of Primary Murine Microglia Cells

Authors: Xuqin Chen
Xuqin ChenAffiliation: Department of Neurology, Affiliated Children's Hospital, Soochow University, Suzhou, China
For correspondence: xuqinlili@yahoo.com
Bio-protocol author page: a202
Ye Zhang
Ye ZhangAffiliation: Department of Neurology, Affiliated Children's Hospital, Soochow University, Suzhou, China
Bio-protocol author page: a203
Gaitri Sadadcharam
Gaitri SadadcharamAffiliation: Department of Neurology, Affiliated Children's Hospital, Soochow University, Suzhou, China
Bio-protocol author page: a204
Weili Cui
Weili CuiAffiliation: Department of Neurology, Affiliated Children's Hospital, Soochow University, Suzhou, China
Bio-protocol author page: a205
 and Jiang Huai Wang
Jiang Huai WangAffiliation: Department of Neurology, Affiliated Children's Hospital, Soochow University, Suzhou, China
For correspondence: jh.wang@ucc.ie
Bio-protocol author page: a206
date: 1/5/2013, 10888 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.314.

[Abstract] The following is a detailed protocol for the isolation, purification and culture of murine brain microglia cells using neutral enzyme digestion and shaking. The protocol below is designed to isolate and culture a large number of purified inactivated microglia cells. Neutral enzyme digestion allows ...
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