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Creating a RAW264.7 CRISPR-Cas9 Genome Wide Library

Featured protocol,  Authors: Brooke A Napier
Brooke A Napier Affiliation: Department of Microbiology and Immunology, Stanford School of Medicine, Stanford University, Stanford, CA, USA
Bio-protocol author page: a4566
 and Denise M Monack
Denise M MonackAffiliation: Department of Microbiology and Immunology, Stanford School of Medicine, Stanford University, Stanford, CA, USA
For correspondence: dmonack@stanford.edu
Bio-protocol author page: a4567
date: 5/20/2017, 112 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2320.

Brief version appeared in J Exp Med, Oct 2016
The bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 genome editing tools are used in mammalian cells to knock-out specific genes of interest to elucidate gene function. The CRISPR-Cas9 system requires that the mammalian cell expresses Cas9 endonuclease, guide RNA (gRNA) to lead the endonuclease to the gene of interest, and the PAM sequence that links the Cas9 to the gRNA. CRISPR-Cas9 genome wide libraries are used to screen the effect of each gene in the genome on the cellular phenotype of interest, in an unbiased high-throughput manner. In this protocol, we describe our method of creating a CRISPR-Cas9 genome wide library in a transformed murine macrophage cell-line (RAW264.7). We have employed this library to identify novel mediators in the caspase-11 cell death pathway (Napier et al., 2016); however, this library can then be used to screen the importance of specific genes in multiple murine macrophage cellular pathways.

Detection of ASC Oligomerization by Western Blotting

Featured protocol,  Authors: Jérôme Lugrin
Jérôme LugrinAffiliation: Department of Biochemistry, University of Lausanne, CH-1066 Epalinges, Switzerland
Bio-protocol author page: a4538
 and Fabio Martinon
Fabio Martinon Affiliation: Department of Biochemistry, University of Lausanne, CH-1066 Epalinges, Switzerland
For correspondence: Fabio.Martinon@unil.ch
Bio-protocol author page: a4539
date: 5/20/2017, 95 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2292.

Brief version appeared in Proc Natl Acad Sci U S A, Aug 2016
The apoptosis-associated speck-like protein with a caspase-recruitment domain (ASC) adaptor protein bridges inflammasome sensors and caspase-1. Upon inflammasome activation, ASC nucleates in a prion-like manner into a large and single platform responsible for the recruitment and the activation of caspase-1. Active caspase-1 will in turn promote the proteolytic maturation of the pro-inflammatory cytokine IL-1β. ASC oligomerization is a direct evidence for inflammasome activation and its detection allows a read-out independent of caspase-1 and IL-1β. This protocol describes how to detect the oligomerization of ASC by Western blot.

Lung Section Staining and Microscopy

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: 5/20/2017, 107 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2286.

Brief version appeared in Mucosal Immunol, May 2016
Our protocol describes immunofluorescent staining, hematoxylin and eosin staining and Masson’s trichrome staining on lung sections.

Efficient Production of Functional Human NKT Cells from Induced Pluripotent Stem Cells − Reprogramming of Human Vα24+iNKT Cells

Featured protocol,  Authors: Daisuke Yamada
Daisuke YamadaAffiliation: Laboratory for Developmental Genetics, Center for Integrative Medical Sciences (IMS), RIKEN, Yokohama, Japan
For correspondence: daisuke.yamada@riken.jp
Bio-protocol author page: a4503
Tomonori Iyoda
Tomonori IyodaAffiliation: Laboratory for Immunotherapy, Center for Integrative Medical Sciences (IMS), RIKEN, Yokohama, Japan
Bio-protocol author page: a4497
Kanako Shimizu
Kanako ShimizuAffiliation: Laboratory for Immunotherapy, Center for Integrative Medical Sciences (IMS), RIKEN, Yokohama, Japan
Bio-protocol author page: a4498
Yusuke Sato
Yusuke SatoAffiliation: Laboratory for Immunotherapy, Center for Integrative Medical Sciences (IMS), RIKEN, Yokohama, Japan
Bio-protocol author page: a4501
Haruhiko Koseki
Haruhiko KosekiAffiliation: Laboratory for Developmental Genetics, Center for Integrative Medical Sciences (IMS), RIKEN, Yokohama, Japan
Bio-protocol author page: a4502
 and Shin-ichiro Fujii
Shin-ichiro FujiiAffiliation: Laboratory for Immunotherapy, Center for Integrative Medical Sciences (IMS), RIKEN, Yokohama, Japan
For correspondence: shin-ichiro.fujii@riken.jp
Bio-protocol author page: a4504
date: 5/20/2017, 93 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2277.

Brief version appeared in Stem Cells, Dec 2016
Antigen-specific T cell-derived induced pluripotent stem cells (iPSCs) have been shown to re-differentiate into functional T cells and thus provide a potential source of T cells that could be useful for cancer immunotherapy. Human Vα24+ invariant natural killer T (Vα24+iNKT) cells are subset of T cells that are characterized by the expression of an invariant Vα24-Jα18 paired with Vβ11, that recognize glycolipids, such as α-galactosylceramide (α-GalCer), presented by the MHC class I-like molecule CD1d. Vα24+iNKT cells capable of producing IFN-γ are reported to augment anti-tumor responses, which affects both NK cells and CD8+ cytotoxic T lymphocytes to eliminate MHC- and MHC+ tumor cells, respectively. Here we describe a robust protocol to reprogram human Vα24+iNKT cells into iPSC, and then to re-differentiate them into Vα24+iNKT cells (iPS-Vα24+iNKT). We further provide a protocol to measure the activity of iPS-Vα24+iNKT cells.

Endogenous C-terminal Tagging by CRISPR/Cas9 in Trypanosoma cruzi

Featured protocol,  Authors: Noelia Lander*
Noelia LanderAffiliation: Departamento de Patologia Clínica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Campinas, São Paulo, 13083, Brazil
For correspondence: noelia@uga.edu
Bio-protocol author page: a4559
Miguel A. Chiurillo*
Miguel A. ChiurilloAffiliation: Departamento de Patologia Clínica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Campinas, São Paulo, 13083, Brazil
Bio-protocol author page: a4560
Aníbal E. Vercesi
Aníbal E. VercesiAffiliation: Departamento de Patologia Clínica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Campinas, São Paulo, 13083, Brazil
Bio-protocol author page: a4561
 and Roberto Docampo
Roberto DocampoAffiliation 1: Departamento de Patologia Clínica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Campinas, São Paulo, 13083, Brazil
Affiliation 2: Center for Tropical and Emerging Global Diseases and Department of Cellular Biology, University of Georgia, Athens, Georgia, USA
Bio-protocol author page: a4562
 (*contributed equally to this work) date: 5/20/2017, 113 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2299.

Brief version appeared in J Biol Chem, Dec 2016
To achieve the C-terminal tagging of endogenous proteins in T. cruzi we use the Cas9/pTREX-n vector (Lander et al., 2015) to insert a specific tag sequence (3xHA or 3xc-Myc) at the 3’ end of a specific gene of interest (GOI). Chimeric sgRNA targeting the 3’ end of the GOI is PCR-amplified and cloned into Cas9/pTREX-n vector. Then a DNA donor molecule to induce DNA repair by homologous recombination is amplified. This donor sequence contains the tag sequence and a marker for antibiotic resistance, plus 100 bp homology arms corresponding to regions located right upstream of the stop codon and downstream of the Cas9 target site at the GOI locus. Vectors pMOTag23M (Oberholzer et al., 2006) or pMOHX1Tag4H (Lander et al., 2016b) are used as PCR templates for DNA donor amplification. Epimastigotes co-transfected with the sgRNA/Cas9/pTREX-n construct and the DNA donor cassette are then cultured for 5 weeks with antibiotics for selection of double resistant parasites. Endogenous gene tagging is finally verified by PCR and Western blot analysis.

Escherichia coli Infection of Drosophila

Featured protocol,  Authors: Charles Tracy
Charles TracyAffiliation: Department of Neuroscience, University of Texas Southwestern Medical Center, Dallas, USA
Bio-protocol author page: a4450
 and Helmut Krämer
Helmut KrämerAffiliation 1: Department of Neuroscience, University of Texas Southwestern Medical Center, Dallas, USA
Affiliation 2: Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, USA
For correspondence: helmut.kramer@utsouthwestern.edu
Bio-protocol author page: a4451
date: 5/5/2017, 170 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2256.

Brief version appeared in Immunity, Aug 2016
Following septic insults, healthy insects, just like vertebrates, mount a complex immune response to contain and destroy pathogens. The failure to efficiently clear bacterial infections in immuno-compromised fly mutants leads to higher mortality rates which provide a powerful indicator for genes with important roles in innate immunity. The following protocol is designed to reproducibly inject a known amount of non-pathogenic E. coli into otherwise sterile flies and to measure the survival of flies after infection. The protocol can be easily adapted to different types of bacteria.

Analysis of Mitochondrial Transfer in Direct Co-cultures of Human Monocyte-derived Macrophages (MDM) and Mesenchymal Stem Cells (MSC)

Featured protocol,  Authors: Megan V. Jackson
Megan V. JacksonAffiliation: Centre for Experimental Medicine, School of Medicine, Dentistry and Biomedical Sciences, Queen’s University Belfast, Belfast , Northern Ireland
Bio-protocol author page: a4447
 and Anna D. Krasnodembskaya
Anna D. KrasnodembskayaAffiliation: Centre for Experimental Medicine, School of Medicine, Dentistry and Biomedical Sciences, Queen’s University Belfast, Belfast , Northern Ireland
For correspondence: a.krasnodembskaya@qub.ac.uk
Bio-protocol author page: a4449
date: 5/5/2017, 189 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2255.

Brief version appeared in Stem Cells, Aug 2016
Mesenchymal stem/stromal cells (MSC) are adult stem cells which have been shown to improve survival, enhance bacterial clearance and alleviate inflammation in pre-clinical models of acute respiratory distress syndrome (ARDS) and sepsis. These diseases are characterised by uncontrolled inflammation often underpinned by bacterial infection. The mechanisms of MSC immunomodulatory effects are not fully understood yet. We sought to investigate MSC cell contact-dependent communication with alveolar macrophages (AM), professional phagocytes which play an important role in the lung inflammatory responses and anti-bacterial defence. With the use of a basic direct co-culture system, confocal microscopy and flow cytometry we visualised and effectively quantified MSC mitochondrial transfer to AM through tunnelling nanotubes (TNT). To model the human AM, primary monocytes were isolated from human donor blood and differentiated into macrophages (monocyte derived macrophages, MDM) in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF), thus allowing adaptation of an AM-like phenotype (de Almeida et al., 2000; Guilliams et al., 2013). Human bone-marrow derived MSC, were labelled with mitochondria-specific fluorescent stain, washed extensively, seeded into the tissue culture plate with MDMs at the ratio of 1:20 (MSC/MDM) and co-cultured for 24 h. TNT formation and mitochondrial transfer were visualised by confocal microscopy and semi-quantified by flow cytometry. By using the method we described here we established that MSC use TNTs as the means to transfer mitochondria to macrophages. Further studies demonstrated that mitochondrial transfer enhances macrophage oxidative phosphorylation and phagocytosis. When TNT formation was blocked by cytochalasin B, MSC effect on macrophage phagocytosis was completely abrogated. This is the first study to demonstrate TNT-mediated mitochondrial transfer from MSC to innate immune cells.

Creating a RAW264.7 CRISPR-Cas9 Genome Wide Library

Authors: Brooke A Napier
Brooke A Napier Affiliation: Department of Microbiology and Immunology, Stanford School of Medicine, Stanford University, Stanford, CA, USA
Bio-protocol author page: a4566
 and Denise M Monack
Denise M MonackAffiliation: Department of Microbiology and Immunology, Stanford School of Medicine, Stanford University, Stanford, CA, USA
For correspondence: dmonack@stanford.edu
Bio-protocol author page: a4567
date: 5/20/2017, 112 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2320.

[Abstract] The bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 genome editing tools are used in mammalian cells to knock-out specific genes of interest to elucidate gene function. The CRISPR-Cas9 system requires that the mammalian cell expresses Cas9 endonuclease, guide RNA (gRNA) to lead the endonuclease to the gene of interest, ...

Detection of ASC Oligomerization by Western Blotting

Authors: Jérôme Lugrin
Jérôme LugrinAffiliation: Department of Biochemistry, University of Lausanne, CH-1066 Epalinges, Switzerland
Bio-protocol author page: a4538
 and Fabio Martinon
Fabio Martinon Affiliation: Department of Biochemistry, University of Lausanne, CH-1066 Epalinges, Switzerland
For correspondence: Fabio.Martinon@unil.ch
Bio-protocol author page: a4539
date: 5/20/2017, 95 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2292.

[Abstract] The apoptosis-associated speck-like protein with a caspase-recruitment domain (ASC) adaptor protein bridges inflammasome sensors and caspase-1. Upon inflammasome activation, ASC nucleates in a prion-like manner into a large and single platform responsible for the recruitment and the activation of caspase-1. Active caspase-1 will in turn promote the ...

Lung Section Staining and Microscopy

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: 5/20/2017, 107 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2286.

[Abstract] Our protocol describes immunofluorescent staining, hematoxylin and eosin staining and Masson’s trichrome staining on lung sections....

Efficient Production of Functional Human NKT Cells from Induced Pluripotent Stem Cells − Reprogramming of Human Vα24+iNKT Cells

Authors: Daisuke Yamada
Daisuke YamadaAffiliation: Laboratory for Developmental Genetics, Center for Integrative Medical Sciences (IMS), RIKEN, Yokohama, Japan
For correspondence: daisuke.yamada@riken.jp
Bio-protocol author page: a4503
Tomonori Iyoda
Tomonori IyodaAffiliation: Laboratory for Immunotherapy, Center for Integrative Medical Sciences (IMS), RIKEN, Yokohama, Japan
Bio-protocol author page: a4497
Kanako Shimizu
Kanako ShimizuAffiliation: Laboratory for Immunotherapy, Center for Integrative Medical Sciences (IMS), RIKEN, Yokohama, Japan
Bio-protocol author page: a4498
Yusuke Sato
Yusuke SatoAffiliation: Laboratory for Immunotherapy, Center for Integrative Medical Sciences (IMS), RIKEN, Yokohama, Japan
Bio-protocol author page: a4501
Haruhiko Koseki
Haruhiko KosekiAffiliation: Laboratory for Developmental Genetics, Center for Integrative Medical Sciences (IMS), RIKEN, Yokohama, Japan
Bio-protocol author page: a4502
 and Shin-ichiro Fujii
Shin-ichiro FujiiAffiliation: Laboratory for Immunotherapy, Center for Integrative Medical Sciences (IMS), RIKEN, Yokohama, Japan
For correspondence: shin-ichiro.fujii@riken.jp
Bio-protocol author page: a4504
date: 5/20/2017, 93 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2277.

[Abstract] Antigen-specific T cell-derived induced pluripotent stem cells (iPSCs) have been shown to re-differentiate into functional T cells and thus provide a potential source of T cells that could be useful for cancer immunotherapy. Human Vα24+ invariant natural killer T (Vα24+iNKT) cells are subset of T cells that are characterized by the expression of an ...

Endogenous C-terminal Tagging by CRISPR/Cas9 in Trypanosoma cruzi

Authors: Noelia Lander*
Noelia LanderAffiliation: Departamento de Patologia Clínica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Campinas, São Paulo, 13083, Brazil
For correspondence: noelia@uga.edu
Bio-protocol author page: a4559
Miguel A. Chiurillo*
Miguel A. ChiurilloAffiliation: Departamento de Patologia Clínica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Campinas, São Paulo, 13083, Brazil
Bio-protocol author page: a4560
Aníbal E. Vercesi
Aníbal E. VercesiAffiliation: Departamento de Patologia Clínica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Campinas, São Paulo, 13083, Brazil
Bio-protocol author page: a4561
 and Roberto Docampo
Roberto DocampoAffiliation 1: Departamento de Patologia Clínica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Campinas, São Paulo, 13083, Brazil
Affiliation 2: Center for Tropical and Emerging Global Diseases and Department of Cellular Biology, University of Georgia, Athens, Georgia, USA
Bio-protocol author page: a4562
 (*contributed equally to this work) date: 5/20/2017, 113 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2299.

[Abstract] To achieve the C-terminal tagging of endogenous proteins in T. cruzi we use the Cas9/pTREX-n vector (Lander et al., 2015) to insert a specific tag sequence (3xHA or 3xc-Myc) at the 3’ end of a specific gene of interest (GOI). Chimeric sgRNA targeting the 3’ end of the GOI is PCR-amplified and cloned into Cas9/pTREX-n vector. Then a DNA donor molecule ...

Escherichia coli Infection of Drosophila

Authors: Charles Tracy
Charles TracyAffiliation: Department of Neuroscience, University of Texas Southwestern Medical Center, Dallas, USA
Bio-protocol author page: a4450
 and Helmut Krämer
Helmut KrämerAffiliation 1: Department of Neuroscience, University of Texas Southwestern Medical Center, Dallas, USA
Affiliation 2: Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, USA
For correspondence: helmut.kramer@utsouthwestern.edu
Bio-protocol author page: a4451
date: 5/5/2017, 170 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2256.

[Abstract] Following septic insults, healthy insects, just like vertebrates, mount a complex immune response to contain and destroy pathogens. The failure to efficiently clear bacterial infections in immuno-compromised fly mutants leads to higher mortality rates which provide a powerful indicator for genes with important roles in innate immunity. The following ...

Analysis of Mitochondrial Transfer in Direct Co-cultures of Human Monocyte-derived Macrophages (MDM) and Mesenchymal Stem Cells (MSC)

Authors: Megan V. Jackson
Megan V. JacksonAffiliation: Centre for Experimental Medicine, School of Medicine, Dentistry and Biomedical Sciences, Queen’s University Belfast, Belfast , Northern Ireland
Bio-protocol author page: a4447
 and Anna D. Krasnodembskaya
Anna D. KrasnodembskayaAffiliation: Centre for Experimental Medicine, School of Medicine, Dentistry and Biomedical Sciences, Queen’s University Belfast, Belfast , Northern Ireland
For correspondence: a.krasnodembskaya@qub.ac.uk
Bio-protocol author page: a4449
date: 5/5/2017, 189 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2255.

[Abstract] Mesenchymal stem/stromal cells (MSC) are adult stem cells which have been shown to improve survival, enhance bacterial clearance and alleviate inflammation in pre-clinical models of acute respiratory distress syndrome (ARDS) and sepsis. These diseases are characterised by uncontrolled inflammation often underpinned by bacterial infection. The mechanisms ...

Lentiviral Barcode Labeling and Transplantation of Fetal Liver Hematopoietic Stem and Progenitor Cells

Authors: Trine A. Kristiansen
Trine A. KristiansenAffiliation: Division of Molecular Hematology, Department of Laboratory Medicine, Lund Stem Cell Center, Faculty of Medicine, Lund University, Lund, Sweden
Bio-protocol author page: a4393
Alexander Doyle
Alexander Doyle Affiliation: Division of Molecular Hematology, Department of Laboratory Medicine, Lund Stem Cell Center, Faculty of Medicine, Lund University, Lund, Sweden
Bio-protocol author page: a4394
 and Joan Yuan
Joan YuanAffiliation: Division of Molecular Hematology, Department of Laboratory Medicine, Lund Stem Cell Center, Faculty of Medicine, Lund University, Lund, Sweden
For correspondence: joan.yuan@med.lu.se
Bio-protocol author page: a4395
date: 4/20/2017, 229 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2242.

[Abstract] Cellular barcoding enables the dissection of clonal dynamics in heterogeneous cell populations through single cell lineage tracing. The labeling of hematopoietic stem and progenitor cells (HSPCs) with unique and heritable DNA barcodes, makes it possible to resolve donor cell heterogeneity in terms of differentiation potential and lineage bias at the ...

Screening for Novel Endogenous Inflammatory Stimuli Using the Secreted Embryonic Alkaline Phosphatase NF-κB Reporter Assay

Authors: Lorena Zuliani-Alvarez
Lorena Zuliani-AlvarezAffiliation: Kennedy Institute of Rheumatology, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Roosevelt Drive, Headington, Oxford, OX3 7FY, UK
Bio-protocol author page: a4338
Anna M. Piccinini
Anna M. PiccininiAffiliation: Kennedy Institute of Rheumatology, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Roosevelt Drive, Headington, Oxford, OX3 7FY, UK
Present address: School of Pharmacy, University of Nottingham, Nottingham, NG7 2RD, UK
Bio-protocol author page: a4339
 and Kim S Midwood
Kim S MidwoodAffiliation: Kennedy Institute of Rheumatology, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Roosevelt Drive, Headington, Oxford, OX3 7FY, UK
For correspondence: kim.midwood@kennedy.ox.ac.uk
Bio-protocol author page: a4340
date: 4/5/2017, 304 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2220.

[Abstract] An immune response can be activated by pathogenic stimuli, as well as endogenous danger signals, triggering the activation of pattern recognition receptors and initiating signalling cascades that lead to inflammation. This method uses THP1-BlueTM cells, a human monocytic cell line which contains an embryonic alkaline phosphatase reporter gene allowing ...

In vitro Detection of Neutrophil Traps and Post-attack Cell Wall Changes in Candida Hyphae

Authors: Alex Hopke
Alex HopkeAffiliation: Molecular & Biomedical Sciences, University of Maine, Orono, Maine, USA
Bio-protocol author page: a4321
 and Robert T. Wheeler
Robert T. WheelerAffiliation 1: Molecular & Biomedical Sciences, University of Maine, Orono, Maine, USA
Affiliation 2: Graduate School of Biomedical Sciences and Engineering, University of Maine, Orono, Maine, USA
For correspondence: robert.wheeler1@maine.edu
Bio-protocol author page: a4322
date: 4/5/2017, 297 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2213.

[Abstract] In this protocol we describe how to visualize neutrophil extracellular traps (NETs) and fungal cell wall changes in the context of the coculture of mouse neutrophils with fungal hyphae of Candida albicans. These protocols are easily adjusted to test a wide array of hypotheses related to the impact of immune cells on fungi and the cell wall, making ...
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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, 43493 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, 35601 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, 31126 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, 22265 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, 21844 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, 20133 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, 18067 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, 17740 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, 13186 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, 13185 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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