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Bioelectrospray Methodology for Dissection of the Host-pathogen Interaction in Human Tuberculosis

Featured protocol,  Authors: Liku B Tezera
Liku B TezeraAffiliation: Clinical and Experimental Sciences, University of Southampton, Southampton, United Kingdom
Bio-protocol author page: a4926
Magdalena K Bielecka
Magdalena K Bielecka Affiliation: Clinical and Experimental Sciences, University of Southampton, Southampton, United Kingdom
Bio-protocol author page: a4927
 and Paul T Elkington
Paul T ElkingtonAffiliation: Clinical and Experimental Sciences, University of Southampton, Southampton, United Kingdom
For correspondence: p.elkington@soton.ac.uk
Bio-protocol author page: a4925
date: 7/20/2017, 27 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2418.

Brief version appeared in eLife, Jan 2017
Standard cell culture models have been used to investigate disease pathology and to test new therapies for over fifty years. However, these model systems have often failed to mimic the changes occurring within three-dimensional (3-D) space where pathology occurs in vivo. To truthfully represent this, an emerging paradigm in biology is the importance of modelling disease in a physiologically relevant 3-D environment. One of the approaches for 3-D cell culture is bioelectrospray technology. This technique uses an alginate-based 3-D environment as an inert backbone within which mammalian cells and extracellular matrix can be incorporated. These alginate-based matrices produce highly reproducible results and can be mixed with different extracellular matrix components. This protocol describes a 3-D system incorporating mycobacteria, primary human blood mononuclear cells and collagen-alginate matrix to dissect the host-pathogen interaction in tuberculosis.

Preparation of Mosquito Salivary Gland Extract and Intradermal Inoculation of Mice

Featured protocol,  Authors: Michael A. Schmid
Michael A. SchmidAffiliation: Rega Institute for Medical Research, Virology and Chemotherapy, Department of Immunology and Microbiology, University of Leuven, Leuven, Belgium
For correspondence: michael.alex.schmid@gmail.com
Bio-protocol author page: a4893
Elizabeth Kauffman
Elizabeth KauffmanAffiliation: Wadsworth Center, New York State Department of Health, Albany, New York, USA
Bio-protocol author page: a4894
Anne Payne
Anne PayneAffiliation: Wadsworth Center, New York State Department of Health, Albany, New York, USA
Bio-protocol author page: a4895
Eva Harris
Eva HarrisAffiliation: Division of Infectious Diseases and Vaccinology, School of Public Health, University of California, Berkeley, California, USA
Bio-protocol author page: a4896
 and Laura D. Kramer
Laura D. KramerAffiliation 1: Wadsworth Center, New York State Department of Health, Albany, New York, USA
Affiliation 2: School of Public Health, State University of New York at Albany, Albany, New York, USA
For correspondence: laura.kramer@health.ny.gov
Bio-protocol author page: a4897
date: 7/20/2017, 29 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2407.

Brief version appeared in PLoS Pathog, Jun 2016
Mosquito-transmitted pathogens are among the leading causes of severe disease and death in humans. Components within the saliva of mosquito vectors facilitate blood feeding, modulate host responses, and allow efficient transmission of pathogens, such as Dengue, Zika, yellow fever, West Nile, Japanese encephalitis, and chikungunya viruses, as well as Plasmodium parasites, among others. Here, we describe standardized methods to assess the impact of mosquito-derived factors on immune responses and pathogenesis in mouse models of infection. This protocol includes the generation of mosquito salivary gland extracts and intradermal inoculation of mouse ears. Ultimately, the information obtained from using these techniques can help reveal fundamental mechanisms of interaction between pathogens, mosquito vectors, and the mammalian host. In addition, this protocol can help establish improved infection challenge models for pre-clinical testing of vaccines or therapeutics that take into account the natural route of transmission via mosquitoes.

Analyzing the Properties of Murine Intestinal Mucins by Electrophoresis and Histology

Featured protocol,  Authors: Ran Wang
Ran WangAffiliation: Inflammatory Disease Biology and Therapeutics Group, Mater Research Institute–The University of Queensland, Translational Research Institute, Brisbane, Australia
Bio-protocol author page: a4833
 and Sumaira Z. Hasnain
Sumaira Z. HasnainAffiliation: Inflammatory Disease Biology and Therapeutics Group, Mater Research Institute–The University of Queensland, Translational Research Institute, Brisbane, Australia
For correspondence: sumaira.hasnain@mater.uq.edu.au
Bio-protocol author page: a4834
date: 7/20/2017, 20 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2394.

Brief version appeared in PLoS Pathog, Feb 2017
Specialized secretory cells known as goblet cells in the intestine and respiratory epithelium are responsible for the secretion of mucins. Mucins are large heavily glycosylated proteins and typically have a molecular mass higher than 106 Da. These large proteins are densely substituted with short glycan chains, which have many important functional roles including determining the hydration and viscoelastic properties of the mucus gel that lines and protects the intestinal epithelium. In this protocol, we comprehensively describe the method for extraction of murine mucus and its analysis by agarose gel electrophoresis. Additionally we describe the use of High Iron Diamine-Alcian Blue, Periodic Acid Schiff’s-Alcian Blue and immune–staining methods to identify and differentiate between the different states of glycosylation on these mucin glycoproteins, in particular with a focus on sulphation and sialylation.

Vaginal HSV-2 Infection and Tissue Analysis

Featured protocol,  Authors: Marie Beck Iversen
Marie Beck IversenAffiliation: Department of Biomedicine, Faculty of Health Sciences, Aarhus University, Aarhus, Denmark
Bio-protocol author page: a4818
Søren Riis Paludan
Søren Riis PaludanAffiliation: Department of Biomedicine, Faculty of Health Sciences, Aarhus University, Aarhus, Denmark
Bio-protocol author page: a4819
 and Christian Kanstrup Holm
Christian Kanstrup HolmAffiliation: Department of Biomedicine, Faculty of Health Sciences, Aarhus University, Aarhus, Denmark
For correspondence: holm@biomed.au.dk
Bio-protocol author page: a4820
date: 7/5/2017, 188 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2383.

Brief version appeared in Nat Immunol, Feb 2016
The vaginal murine HSV-2 infection model is very useful in studying mucosal immunity against HSV-2 (Overall et al., 1975; Renis et al., 1976; Parr and Parr, 2003). Histologically, the vagina of Depo-Provera-treated mice is lined by a single layer of mucus secreting columnar epithelial cells overlying two to three layers of proliferative cells. Even though this is morphologically different from the human vagina, it closely resembles the endocervical epithelium, which is thought to be the primary site of HSV-2 infection in women (Parr et al., 1994; Kaushic et al., 2011). In the protocol presented here, mice are pre-treated with Depo-Provera before intra-vaginal inoculation with HSV-2. The virus replicates in the mucosal epithelium from where it spreads to and replicates in the CNS including the spinal cord, brain stem, cerebrum and cerebellum. Cytokine responses can be detected in vaginal washings using ELISA or in vaginal tissues using qPCR. Further, the recruitment of leukocytes to the vagina can be determined by flow cytometry. The model is suitable for research of both innate and adaptive immunity to HSV-2 infection.

Thermal Stability of Heterotrimeric pMHC Proteins as Determined by Circular Dichroism Spectroscopy

Featured protocol,  Authors: Anna Fuller
Anna FullerAffiliation: Division of Infection and Immunity and Systems Immunity Research Institute, Cardiff University School of Medicine, Heath Park, Cardiff, UK
Bio-protocol author page: a4810
Aaron Wall
Aaron WallAffiliation: Division of Infection and Immunity and Systems Immunity Research Institute, Cardiff University School of Medicine, Heath Park, Cardiff, UK
Bio-protocol author page: a4811
Michael D Crowther
Michael D CrowtherAffiliation: Division of Infection and Immunity and Systems Immunity Research Institute, Cardiff University School of Medicine, Heath Park, Cardiff, UK
Bio-protocol author page: a4812
Angharad Lloyd
Angharad LloydAffiliation: Division of Infection and Immunity and Systems Immunity Research Institute, Cardiff University School of Medicine, Heath Park, Cardiff, UK
Bio-protocol author page: a4813
Alexei Zhurov
Alexei ZhurovAffiliation: Cardiff University School of Dentistry, Heath Park, Cardiff, UK
Bio-protocol author page: a4814
Andrew K. Sewell
Andrew K. SewellAffiliation: Division of Infection and Immunity and Systems Immunity Research Institute, Cardiff University School of Medicine, Heath Park, Cardiff, UK
Bio-protocol author page: a4815
David K. Cole
David K. ColeAffiliation: Division of Infection and Immunity and Systems Immunity Research Institute, Cardiff University School of Medicine, Heath Park, Cardiff, UK
For correspondence: ColeDK@cf.ac.uk
Bio-protocol author page: a4816
 and Konrad Beck
Konrad BeckAffiliation: Cardiff University School of Dentistry, Heath Park, Cardiff, UK
For correspondence: BeckK@cf.ac.uk
Bio-protocol author page: a4799
date: 7/5/2017, 205 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2366.

Brief version appeared in J Clin Invest, Jun 2016
T cell receptor (TCR) recognition of foreign peptide fragments, presented by peptide major histocompatibility complex (pMHC), governs T-cell mediated protection against pathogens and cancer. Many factors govern T-cell sensitivity, including the affinity of the TCR-pMHC interaction and the stability of pMHC on the surface of antigen presenting cells. These factors are particularly relevant for the peptide vaccination field, in which more stable pMHC interactions could enable more effective protection against disease. Here, we discuss a method for the determination of pMHC stability that we have used to investigate HIV immune escape, T-cell sensitivity to cancer antigens and mechanisms leading to autoimmunity.

Qualitative and Quantitative Assay for Detection of Circulating Autoantibodies against Human Aortic Antigen

Featured protocol,  Authors: Brent Veerman
Brent VeermanAffiliation: Department of Surgery, University of Toledo College of Medicine and Life Sciences, Toledo, USA
Bio-protocol author page: a4752
 and Ritu Chakravarti
Ritu ChakravartiAffiliation: Department of Surgery, University of Toledo College of Medicine and Life Sciences, Toledo, USA
For correspondence: ritu.chakravarti@utoledo.edu
Bio-protocol author page: a3737
date: 7/5/2017, 189 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2367.

Brief version appeared in Arthritis Rheumatol, Jul 2015
Increased amount of autoantibodies in human sera are the hallmark of autoimmune diseases (Wang et al., 2015). In case of known antigen, detection of autoantibodies is done using laboratory based methods. However, in most autoimmune diseases, knowledge of self-antigen is still vague. We have developed an ELISA-based quantitative assay to detect the presence of autoantibodies as well as to measure the circulating autoantibodies in the sera of patients suffering with large vessel vasculitis (LVV), an autoimmune disease (Chakravarti et al., 2015). Using this assay, we detected the increase in anti-aortic antibodies in LVV patient’s sera. We have further verified the results by independent biochemical techniques and found the specificity to be > 94% (Chakravarti et al., 2015). This method can be uniquely modified to suit any autoimmune, in particular organ specific, disease and thus has wider applications in the detection and quantification of autoantibodies.

EAE Induction by Passive Transfer of MOG-specific CD4+ T Cells

Featured protocol,  Authors: Yuki Tanaka*
Yuki TanakaAffiliation: Division of Molecular Neuroimmunology, Institute for Genetic Medicine and Graduate School of Medicine, Hokkaido University, Sapporo, Japan
Bio-protocol author page: a4755
Yasunobu Arima*
Yasunobu ArimaAffiliation: Division of Molecular Neuroimmunology, Institute for Genetic Medicine and Graduate School of Medicine, Hokkaido University, Sapporo, Japan
Bio-protocol author page: a4756
Kotaro Higuchi
Kotaro HiguchiAffiliation: Division of Molecular Neuroimmunology, Institute for Genetic Medicine and Graduate School of Medicine, Hokkaido University, Sapporo, Japan
Bio-protocol author page: a4757
Takuto Ohki
Takuto OhkiAffiliation: Division of Molecular Neuroimmunology, Institute for Genetic Medicine and Graduate School of Medicine, Hokkaido University, Sapporo, Japan
Bio-protocol author page: a4758
Mohamed Elfeky
Mohamed ElfekyAffiliation: Division of Molecular Neuroimmunology, Institute for Genetic Medicine and Graduate School of Medicine, Hokkaido University, Sapporo, Japan
Bio-protocol author page: a4759
Mitsutoshi Ota
Mitsutoshi OtaAffiliation: Division of Molecular Neuroimmunology, Institute for Genetic Medicine and Graduate School of Medicine, Hokkaido University, Sapporo, Japan
Bio-protocol author page: a4760
Daisuke Kamimura
Daisuke KamimuraAffiliation: Division of Molecular Neuroimmunology, Institute for Genetic Medicine and Graduate School of Medicine, Hokkaido University, Sapporo, Japan
For correspondence: kamimura@igm.hokudai.ac.jp
Bio-protocol author page: a4761
 and Masaaki Murakami
Masaaki MurakamiAffiliation: Division of Molecular Neuroimmunology, Institute for Genetic Medicine and Graduate School of Medicine, Hokkaido University, Sapporo, Japan
For correspondence: murakami@igm.hokudai.ac.jp
Bio-protocol author page: a4762
 (*contributed equally to this work) date: 7/5/2017, 203 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2370.

Brief version appeared in Elife, Aug 2015
Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis (MS), which is a chronic inflammatory disease of the central nervous system (CNS). It is characterized by focal demyelination and inflammatory responses mediated by myelin-specific autoreactive CD4+ T cells. Using a passive transfer model of EAE in mice, we have demonstrated that regional specific neural signals by sensory-sympathetic communications create gateways for immune cells at specific blood vessels of the CNS, a phenomenon known as the gateway reflex (Arima et al., 2012; Tracey, 2012; Arima et al., 2013; Sabharwal et al., 2014; Arima et al., 2015b). Here we describe protocols for passive transfer model of EAE using freshly isolated (MOG)-specific CD4+ T cells or periodically restimulated MOG-specific CD4+ T cell lines, which are suitable for tracking pathogenic CD4+ T cells in vivo, particularly in the CNS (Ogura et al., 2008; Arima et al., 2012 and 2015b).

In vitro Demonstration and Quantification of Neutrophil Extracellular Trap Formation

Featured protocol,  Authors: Dongsheng Jiang
Dongsheng JiangAffiliation: Department of Dermatology and Allergic Diseases, University of Ulm, Ulm, Germany
Present address: Comprehensive Pneumology Center, Helmholtz Zentrum München, Munich, Germany
For correspondence: dongsheng.jiang@helmholtz-muenchen.de
Bio-protocol author page: a4790
Mona Saffarzadeh
Mona SaffarzadehAffiliation: Department of Biochemistry, School of Medicine, Justus-Liebig-University of Giessen, Giessen, Germany
Present address: Centre for Thrombosis and Haemostasis, University Medical Centre of Mainz, Mainz, Germany
Bio-protocol author page: a4791
 and Karin Scharffetter-Kochanek
Karin Scharffetter-KochanekAffiliation: Department of Dermatology and Allergic Diseases, University of Ulm, Ulm, Germany
For correspondence: karin.scharffetter-kochanek@uniklinik-ulm.de
Bio-protocol author page: a4792
date: 7/5/2017, 176 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2386.

Brief version appeared in Stem Cells, Sep 2016
In the recent decade, neutrophil extracellular traps (NETs) have been identified and confirmed as a new anti-microbial weapon of neutrophils. In this protocol, we describe easy methods to demonstrate NET formation by immunofluorescence staining of extracellular chromatin fiber with anti-DNA/Histone H1 antibody and quantification of NETs by using a non-cell-permeable DNA specific dye Sytox orange.

Bioelectrospray Methodology for Dissection of the Host-pathogen Interaction in Human Tuberculosis

Authors: Liku B Tezera
Liku B TezeraAffiliation: Clinical and Experimental Sciences, University of Southampton, Southampton, United Kingdom
Bio-protocol author page: a4926
Magdalena K Bielecka
Magdalena K Bielecka Affiliation: Clinical and Experimental Sciences, University of Southampton, Southampton, United Kingdom
Bio-protocol author page: a4927
 and Paul T Elkington
Paul T ElkingtonAffiliation: Clinical and Experimental Sciences, University of Southampton, Southampton, United Kingdom
For correspondence: p.elkington@soton.ac.uk
Bio-protocol author page: a4925
date: 7/20/2017, 27 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2418.

[Abstract] Standard cell culture models have been used to investigate disease pathology and to test new therapies for over fifty years. However, these model systems have often failed to mimic the changes occurring within three-dimensional (3-D) space where pathology occurs in vivo. To truthfully represent this, an emerging paradigm in biology is the importance ...

Preparation of Mosquito Salivary Gland Extract and Intradermal Inoculation of Mice

Authors: Michael A. Schmid
Michael A. SchmidAffiliation: Rega Institute for Medical Research, Virology and Chemotherapy, Department of Immunology and Microbiology, University of Leuven, Leuven, Belgium
For correspondence: michael.alex.schmid@gmail.com
Bio-protocol author page: a4893
Elizabeth Kauffman
Elizabeth KauffmanAffiliation: Wadsworth Center, New York State Department of Health, Albany, New York, USA
Bio-protocol author page: a4894
Anne Payne
Anne PayneAffiliation: Wadsworth Center, New York State Department of Health, Albany, New York, USA
Bio-protocol author page: a4895
Eva Harris
Eva HarrisAffiliation: Division of Infectious Diseases and Vaccinology, School of Public Health, University of California, Berkeley, California, USA
Bio-protocol author page: a4896
 and Laura D. Kramer
Laura D. KramerAffiliation 1: Wadsworth Center, New York State Department of Health, Albany, New York, USA
Affiliation 2: School of Public Health, State University of New York at Albany, Albany, New York, USA
For correspondence: laura.kramer@health.ny.gov
Bio-protocol author page: a4897
date: 7/20/2017, 29 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2407.

[Abstract] Mosquito-transmitted pathogens are among the leading causes of severe disease and death in humans. Components within the saliva of mosquito vectors facilitate blood feeding, modulate host responses, and allow efficient transmission of pathogens, such as Dengue, Zika, yellow fever, West Nile, Japanese encephalitis, and chikungunya viruses, as well as ...

Analyzing the Properties of Murine Intestinal Mucins by Electrophoresis and Histology

Authors: Ran Wang
Ran WangAffiliation: Inflammatory Disease Biology and Therapeutics Group, Mater Research Institute–The University of Queensland, Translational Research Institute, Brisbane, Australia
Bio-protocol author page: a4833
 and Sumaira Z. Hasnain
Sumaira Z. HasnainAffiliation: Inflammatory Disease Biology and Therapeutics Group, Mater Research Institute–The University of Queensland, Translational Research Institute, Brisbane, Australia
For correspondence: sumaira.hasnain@mater.uq.edu.au
Bio-protocol author page: a4834
date: 7/20/2017, 20 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2394.

[Abstract] Specialized secretory cells known as goblet cells in the intestine and respiratory epithelium are responsible for the secretion of mucins. Mucins are large heavily glycosylated proteins and typically have a molecular mass higher than 106 Da. These large proteins are densely substituted with short glycan chains, which have many important functional ...

Vaginal HSV-2 Infection and Tissue Analysis

Authors: Marie Beck Iversen
Marie Beck IversenAffiliation: Department of Biomedicine, Faculty of Health Sciences, Aarhus University, Aarhus, Denmark
Bio-protocol author page: a4818
Søren Riis Paludan
Søren Riis PaludanAffiliation: Department of Biomedicine, Faculty of Health Sciences, Aarhus University, Aarhus, Denmark
Bio-protocol author page: a4819
 and Christian Kanstrup Holm
Christian Kanstrup HolmAffiliation: Department of Biomedicine, Faculty of Health Sciences, Aarhus University, Aarhus, Denmark
For correspondence: holm@biomed.au.dk
Bio-protocol author page: a4820
date: 7/5/2017, 188 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2383.

[Abstract] The vaginal murine HSV-2 infection model is very useful in studying mucosal immunity against HSV-2 (Overall et al., 1975; Renis et al., 1976; Parr and Parr, 2003). Histologically, the vagina of Depo-Provera-treated mice is lined by a single layer of mucus secreting columnar epithelial cells overlying two to three layers of proliferative cells. Even ...

Thermal Stability of Heterotrimeric pMHC Proteins as Determined by Circular Dichroism Spectroscopy

Authors: Anna Fuller
Anna FullerAffiliation: Division of Infection and Immunity and Systems Immunity Research Institute, Cardiff University School of Medicine, Heath Park, Cardiff, UK
Bio-protocol author page: a4810
Aaron Wall
Aaron WallAffiliation: Division of Infection and Immunity and Systems Immunity Research Institute, Cardiff University School of Medicine, Heath Park, Cardiff, UK
Bio-protocol author page: a4811
Michael D Crowther
Michael D CrowtherAffiliation: Division of Infection and Immunity and Systems Immunity Research Institute, Cardiff University School of Medicine, Heath Park, Cardiff, UK
Bio-protocol author page: a4812
Angharad Lloyd
Angharad LloydAffiliation: Division of Infection and Immunity and Systems Immunity Research Institute, Cardiff University School of Medicine, Heath Park, Cardiff, UK
Bio-protocol author page: a4813
Alexei Zhurov
Alexei ZhurovAffiliation: Cardiff University School of Dentistry, Heath Park, Cardiff, UK
Bio-protocol author page: a4814
Andrew K. Sewell
Andrew K. SewellAffiliation: Division of Infection and Immunity and Systems Immunity Research Institute, Cardiff University School of Medicine, Heath Park, Cardiff, UK
Bio-protocol author page: a4815
David K. Cole
David K. ColeAffiliation: Division of Infection and Immunity and Systems Immunity Research Institute, Cardiff University School of Medicine, Heath Park, Cardiff, UK
For correspondence: ColeDK@cf.ac.uk
Bio-protocol author page: a4816
 and Konrad Beck
Konrad BeckAffiliation: Cardiff University School of Dentistry, Heath Park, Cardiff, UK
For correspondence: BeckK@cf.ac.uk
Bio-protocol author page: a4799
date: 7/5/2017, 205 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2366.

[Abstract] T cell receptor (TCR) recognition of foreign peptide fragments, presented by peptide major histocompatibility complex (pMHC), governs T-cell mediated protection against pathogens and cancer. Many factors govern T-cell sensitivity, including the affinity of the TCR-pMHC interaction and the stability of pMHC on the surface of antigen presenting cells. ...

Qualitative and Quantitative Assay for Detection of Circulating Autoantibodies against Human Aortic Antigen

Authors: Brent Veerman
Brent VeermanAffiliation: Department of Surgery, University of Toledo College of Medicine and Life Sciences, Toledo, USA
Bio-protocol author page: a4752
 and Ritu Chakravarti
Ritu ChakravartiAffiliation: Department of Surgery, University of Toledo College of Medicine and Life Sciences, Toledo, USA
For correspondence: ritu.chakravarti@utoledo.edu
Bio-protocol author page: a3737
date: 7/5/2017, 189 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2367.

[Abstract] Increased amount of autoantibodies in human sera are the hallmark of autoimmune diseases (Wang et al., 2015). In case of known antigen, detection of autoantibodies is done using laboratory based methods. However, in most autoimmune diseases, knowledge of self-antigen is still vague. We have developed an ELISA-based quantitative assay to detect the ...

EAE Induction by Passive Transfer of MOG-specific CD4+ T Cells

Authors: Yuki Tanaka*
Yuki TanakaAffiliation: Division of Molecular Neuroimmunology, Institute for Genetic Medicine and Graduate School of Medicine, Hokkaido University, Sapporo, Japan
Bio-protocol author page: a4755
Yasunobu Arima*
Yasunobu ArimaAffiliation: Division of Molecular Neuroimmunology, Institute for Genetic Medicine and Graduate School of Medicine, Hokkaido University, Sapporo, Japan
Bio-protocol author page: a4756
Kotaro Higuchi
Kotaro HiguchiAffiliation: Division of Molecular Neuroimmunology, Institute for Genetic Medicine and Graduate School of Medicine, Hokkaido University, Sapporo, Japan
Bio-protocol author page: a4757
Takuto Ohki
Takuto OhkiAffiliation: Division of Molecular Neuroimmunology, Institute for Genetic Medicine and Graduate School of Medicine, Hokkaido University, Sapporo, Japan
Bio-protocol author page: a4758
Mohamed Elfeky
Mohamed ElfekyAffiliation: Division of Molecular Neuroimmunology, Institute for Genetic Medicine and Graduate School of Medicine, Hokkaido University, Sapporo, Japan
Bio-protocol author page: a4759
Mitsutoshi Ota
Mitsutoshi OtaAffiliation: Division of Molecular Neuroimmunology, Institute for Genetic Medicine and Graduate School of Medicine, Hokkaido University, Sapporo, Japan
Bio-protocol author page: a4760
Daisuke Kamimura
Daisuke KamimuraAffiliation: Division of Molecular Neuroimmunology, Institute for Genetic Medicine and Graduate School of Medicine, Hokkaido University, Sapporo, Japan
For correspondence: kamimura@igm.hokudai.ac.jp
Bio-protocol author page: a4761
 and Masaaki Murakami
Masaaki MurakamiAffiliation: Division of Molecular Neuroimmunology, Institute for Genetic Medicine and Graduate School of Medicine, Hokkaido University, Sapporo, Japan
For correspondence: murakami@igm.hokudai.ac.jp
Bio-protocol author page: a4762
 (*contributed equally to this work) date: 7/5/2017, 203 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2370.

[Abstract] Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis (MS), which is a chronic inflammatory disease of the central nervous system (CNS). It is characterized by focal demyelination and inflammatory responses mediated by myelin-specific autoreactive CD4+ T cells. Using a passive transfer model of EAE in mice, we have ...

In vitro Demonstration and Quantification of Neutrophil Extracellular Trap Formation

Authors: Dongsheng Jiang
Dongsheng JiangAffiliation: Department of Dermatology and Allergic Diseases, University of Ulm, Ulm, Germany
Present address: Comprehensive Pneumology Center, Helmholtz Zentrum München, Munich, Germany
For correspondence: dongsheng.jiang@helmholtz-muenchen.de
Bio-protocol author page: a4790
Mona Saffarzadeh
Mona SaffarzadehAffiliation: Department of Biochemistry, School of Medicine, Justus-Liebig-University of Giessen, Giessen, Germany
Present address: Centre for Thrombosis and Haemostasis, University Medical Centre of Mainz, Mainz, Germany
Bio-protocol author page: a4791
 and Karin Scharffetter-Kochanek
Karin Scharffetter-KochanekAffiliation: Department of Dermatology and Allergic Diseases, University of Ulm, Ulm, Germany
For correspondence: karin.scharffetter-kochanek@uniklinik-ulm.de
Bio-protocol author page: a4792
date: 7/5/2017, 176 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2386.

[Abstract] In the recent decade, neutrophil extracellular traps (NETs) have been identified and confirmed as a new anti-microbial weapon of neutrophils. In this protocol, we describe easy methods to demonstrate NET formation by immunofluorescence staining of extracellular chromatin fiber with anti-DNA/Histone H1 antibody and quantification of NETs by using a ...

Functional ex-vivo Imaging of Arterial Cellular Recruitment and Lipid Extravasation

Authors: Emiel P.C. van der Vorst
Emiel P.C. van der VorstAffiliation: Institute for Cardiovascular Prevention (IPEK), Klinikum der Universität München, Ludwig-Maximilians-University, Munich, Germany
For correspondence: Emiel.Van_der_Vorst@med.uni-muenchen.de
Bio-protocol author page: a4681
Sanne L. Maas
Sanne L. MaasAffiliation: Institute for Cardiovascular Prevention (IPEK), Klinikum der Universität München, Ludwig-Maximilians-University, Munich, Germany
Bio-protocol author page: a4682
Almudena Ortega-Gomez
Almudena Ortega-GomezAffiliation: Institute for Cardiovascular Prevention (IPEK), Klinikum der Universität München, Ludwig-Maximilians-University, Munich, Germany
Bio-protocol author page: a4683
Jeroen M.M. Hameleers
Jeroen M.M. HameleersAffiliation: Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, the Netherlands
Bio-protocol author page: a4684
Mariaelvy Bianchini
Mariaelvy BianchiniAffiliation: Institute for Cardiovascular Prevention (IPEK), Klinikum der Universität München, Ludwig-Maximilians-University, Munich, Germany
Bio-protocol author page: a4685
Yaw Asare
Yaw AsareAffiliation: Institute for Stroke and Dementia Research, Klinikum der Universität München, Ludwig-Maximilians-University, Munich, Germany
Bio-protocol author page: a4686
Oliver Soehnlein
Oliver SoehnleinAffiliation 1: Institute for Cardiovascular Prevention (IPEK), Klinikum der Universität München, Ludwig-Maximilians-University, Munich, Germany
Affiliation 2: DZHK (German Centre for Cardiovascular Research), partner site Munich Heart Alliance, Munich, Germany
Affiliation 3: Department of Physiology and Pharmacology (FyFa), Karolinska Institutet, Stockholm, Sweden
Bio-protocol author page: a4687
Yvonne Döring
Yvonne DöringAffiliation: Institute for Cardiovascular Prevention (IPEK), Klinikum der Universität München, Ludwig-Maximilians-University, Munich, Germany
Bio-protocol author page: a4688
Christian Weber
Christian WeberAffiliation 1: Institute for Cardiovascular Prevention (IPEK), Klinikum der Universität München, Ludwig-Maximilians-University, Munich, Germany
Affiliation 2: Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, the Netherlands
Affiliation 3: DZHK (German Centre for Cardiovascular Research), partner site Munich Heart Alliance, Munich, Germany
Bio-protocol author page: a3209
 and Remco T.A. Megens
Remco T.A. MegensAffiliation 1: Institute for Cardiovascular Prevention (IPEK), Klinikum der Universität München, Ludwig-Maximilians-University, Munich, Germany
Affiliation 2: Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, the Netherlands
For correspondence: Remco.Megens@med.uni-muenchen.de
Bio-protocol author page: a4689
date: 6/20/2017, 305 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2344.

[Abstract] The main purpose of this sophisticated and highly versatile method is to visualize and quantify structural vessel wall properties, cellular recruitment, and lipid/dextran extravasation under physiological conditions in living arteries. This will be of interest for a broad range of researchers within the field of inflammation, hypertension, atherosclerosis, ...

ELISPOT Assay to Measure Peptide-specific IFN-γ Production

Authors: Michelle N. Wykes
Michelle N. WykesAffiliation: QIMR Berghofer Medical Research Institute, Brisbane, Australia
For correspondence: michelle.wykes@qimrberghofer.edu.au
Bio-protocol author page: a4571
 and Laurent Renia
Laurent ReniaAffiliation 1: Singapore Immunology Network, Agency for Science, Technology and Research (A*STAR), Singapore, Singapore
Affiliation 2: Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore
For correspondence: renia_laurent@immunol.a-star.edu.sg
Bio-protocol author page: a4572
date: 6/5/2017, 316 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2302.

[Abstract] Interferon-gamma (IFN-γ) is crucial for immunity against intracellular pathogens and for tumor control. It is produced predominantly by natural killer (NK) and natural killer T cells (NKT) as well as by antigen-specific Th1 CD4+ and CD8+ effector T cells. When investigating immune responses against pathogens and cancer cells, measuring antigen-specific ...
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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, 45752 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, 36362 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, 31912 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 ...

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, 23440 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] 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, 22928 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....

[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, 20809 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, 18797 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, 18304 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 ...

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, 14144 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, ...

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, 13674 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 ...
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