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Isolation of PBMCs Using Vacutainer
®
Cellular Preparation Tubes (CPT
TM
)
Authors:
Alaina Puleo
Alaina Puleo
Affiliation:
Institute for Immunity, Transplantation, and Infection, Stanford University School of Medicine, Stanford, Palo Alto, CA, USA
Bio-protocol author page:
a4007
,
Chantia Carroll
Chantia Carroll
Affiliation:
Institute for Immunity, Transplantation, and Infection, Stanford University School of Medicine, Stanford, Palo Alto, CA, USA
Bio-protocol author page:
a4008
,
Holden Maecker
Holden Maecker
Affiliation:
Institute for Immunity, Transplantation, and Infection, Stanford University School of Medicine, Stanford, Palo Alto, CA, USA
For correspondence:
maecker@stanford.edu
Bio-protocol author page:
a1861
and
Rohit Gupta
Rohit Gupta
Affiliation:
Institute for Immunity, Transplantation, and Infection, Stanford University School of Medicine, Stanford, Palo Alto, CA, USA
Bio-protocol author page:
a1867
,
date: 1/20/2017, 1883 views,
0 Q&A
DOI:
https://doi.org/10.21769/BioProtoc.2103
.
[Abstract] Peripheral blood mononuclear cell (PBMC) isolation is commonly done via density gradient centrifugation over Ficoll-Hypaque, a labor-intensive procedure that requires skilled technicians and can contribute to sample variability. Cellular Preparation Tubes (CPTs) are Vacutainer blood draw tubes that contain Ficoll-Hypaque and a gel plug that separates ...
Isolation of Highly Pure Primary Mouse Alveolar Epithelial Type II Cells by Flow Cytometric Cell Sorting
Authors:
Meenal Sinha
Meenal Sinha
Affiliation:
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. Lowell
Affiliation:
Department of Laboratory Medicine and the Program in Immunology, University of California, San Francisco, USA
Bio-protocol author page:
a3724
,
date: 11/20/2016, 1778 views,
0 Q&A
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 10
6
ATII cells per mouse lung. The cell ...
Isolation of Intestinal Mesenchymal Cells from Adult Mice
Authors:
Vasiliki Koliaraki
Vasiliki Koliaraki
Affiliation:
Biomedical Sciences Research Centre (B.S.R.C.) “Alexander Fleming”, Vari, Greece
For correspondence:
koliaraki@fleming.gr
Bio-protocol author page:
a3531
and
George Kollias
George Kollias
Affiliation 1:
Biomedical Sciences Research Centre (B.S.R.C.) “Alexander Fleming”, Vari, Greece
Affiliation 2:
Department of Physiology, Medical School, University of Athens, Athens, Greece
Bio-protocol author page:
a3532
,
date: 9/20/2016, 1669 views,
0 Q&A
DOI:
https://doi.org/10.21769/BioProtoc.1940
.
[Abstract] During the last 20 years intestinal mesenchymal cells (IMCs) have emerged as an important cell type that plays a central role in intestinal development and homeostasis, by providing both structural support and growth regulatory elements. IMCs also actively participate in wound healing responses, thus regulating pathologic conditions such as tissue ...
Isolation of Joint-infiltrating Cells
Authors:
Aoi Akitsu
Aoi Akitsu
Affiliation:
Center for Animal Disease Models, Research Institute for Biomedical Sciences, Tokyo University of Science, Chiba, Japan
For correspondence:
aoipinky@rs.tus.ac.jp
Bio-protocol author page:
a3457
and
Yoichiro Iwakura
Yoichiro Iwakura
Affiliation:
Center for Animal Disease Models, Research Institute for Biomedical Sciences, Tokyo University of Science, Chiba, Japan
Bio-protocol author page:
a3458
,
date: 9/5/2016, 1122 views,
0 Q&A
DOI:
https://doi.org/10.21769/BioProtoc.1911
.
[Abstract] Infiltration of leukocytes into joints is one of the main features of autoimmune inflammatory arthritis. Here, we describe the protocol for isolation of joint-infiltrating cells in mice. This protocol is useful to analyze cell surface antigens and intracellular cytokines by flow cytometry....
Adoptive Transfer of Tumor Expanded Regulatory T Cells (Tregs)
Authors:
Felipe Vences-Catalán
Felipe Vences-Catalán
Affiliation:
Department of Medicine, Division of Oncology, Stanford University, Stanford, CA, USA
For correspondence:
fvences5@stanford.edu
Bio-protocol author page:
a3419
and
Shoshana Levy
Shoshana Levy
Affiliation:
Department of Medicine, Division of Oncology, Stanford University, Stanford, CA, USA
For correspondence:
slevy@stanford.edu
Bio-protocol author page:
a3420
,
date: 8/20/2016, 1498 views,
0 Q&A
DOI:
https://doi.org/10.21769/BioProtoc.1899
.
[Abstract] Regulatory T cells (Tregs), a subset of CD4
+
CD25
+
T cells, infiltrate tumors and suppress antitumor activity of effector T and NK cells. Depletion of Tregs by anti CD25
+
antibodies has been shown to reduce tumor growth and metastasis (Olkhanud
et al
., 2009). Conversely, adoptive transfer of Tregs induced immune suppression and promoted tumor growth ...
Mouse Embryonic Fibroblast Cell Culture and Stimulation
Authors:
Lian-Qun Qiu
Lian-Qun Qiu
Affiliation:
Post-transcriptional Gene Expression Group, Signal Transduction Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, USA
For correspondence:
qiul@niehs.nih.gov
Bio-protocol author page:
a3276
,
Wi S. Lai
Wi S. Lai
Affiliation:
Post-transcriptional Gene Expression Group, Signal Transduction Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, USA
Bio-protocol author page:
a3277
,
Deborah J. Stumpo
Deborah J. Stumpo
Affiliation:
Post-transcriptional Gene Expression Group, Signal Transduction Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, USA
Bio-protocol author page:
a3278
and
Perry J. Blackshear
Perry J. Blackshear
Affiliation:
Post-transcriptional Gene Expression Group, Signal Transduction Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, USA
Bio-protocol author page:
a3279
,
date: 7/5/2016, 2551 views,
0 Q&A
DOI:
https://doi.org/10.21769/BioProtoc.1859
.
[Abstract] Culture of mouse embryonic fibroblast (MEF) cells represents a powerful system to test gene function due to their easy accessibility, rapid growth rates, and the possibility of a large number of experiments. Fibroblasts are a group of heterogeneous resident cells of mesenchymal origin that have various locations, diverse appearances and distinctive ...
Whole-mount Enteroid Proliferation Staining
Authors:
Caitlyn W. Barrett
Caitlyn W. Barrett
Affiliation:
Department of Medicine, Division of Gastroenterology, Hepatology, and Nutrition, and Department of Cancer Biology, Vanderbilt University Medical School, Nashville, USA
Bio-protocol author page:
a3254
,
Sarah P. Short
Sarah P. Short
Affiliation:
Department of Medicine, Division of Gastroenterology, Hepatology, and Nutrition, and Department of Cancer Biology, Vanderbilt University Medical School, Nashville, USA
Bio-protocol author page:
a3255
,
Yash A. Choksi
Yash A. Choksi
Affiliation:
Department of Medicine, Division of Gastroenterology, Hepatology, and Nutrition, and Department of Cancer Biology, Vanderbilt University Medical School, Nashville, USA
Bio-protocol author page:
a3256
and
Christopher Shawn Williams
Christopher Shawn Williams
Affiliation:
Department of Medicine, Division of Gastroenterology, Hepatology, and Nutrition, and Department of Cancer Biology, Vanderbilt University Medical School, Nashville, USA
For correspondence:
Christopher.williams@vanderbilt.edu
Bio-protocol author page:
a3253
,
date: 6/20/2016, 1576 views,
0 Q&A
DOI:
https://doi.org/10.21769/BioProtoc.1837
.
[Abstract] Small intestinal organoids, otherwise known as enteroids, have become an increasingly utilized model for intestinal biology
in vitro
as they recapitulate the various epithelial cells within the intestinal crypt (Mahe
et al.
, 2013; Sato
et al.
, 2009). Assessment of growth dynamics within these cultures is an important step to understanding how alterations ...
Isolation and Culture of the Islets of Langerhans from Mouse Pancreas
Authors:
Kate L Graham
Kate L Graham
Affiliation 1:
Immunology and Diabetes Unit, St. Vincent’s Institute, Melbourne, Australia
Affiliation 2:
Department of Medicine, The University of Melbourne, Melbourne, Australia
Bio-protocol author page:
a3224
,
Stacey Fynch
Stacey Fynch
Affiliation:
Immunology and Diabetes Unit, St. Vincent’s Institute, Melbourne, Australia
Bio-protocol author page:
a3225
,
Evan G Pappas
Evan G Pappas
Affiliation:
Immunology and Diabetes Unit, St. Vincent’s Institute, Melbourne, Australia
Bio-protocol author page:
a3226
,
Christina Tan
Christina Tan
Affiliation:
Immunology and Diabetes Unit, St. Vincent’s Institute, Melbourne, Australia
Bio-protocol author page:
a3227
,
Thomas WH Kay
Thomas WH Kay
Affiliation 1:
Immunology and Diabetes Unit, St. Vincent’s Institute, Melbourne, Australia
Affiliation 2:
Department of Medicine, The University of Melbourne, Melbourne, Australia
Bio-protocol author page:
a3228
and
Helen E Thomas
Helen E Thomas
Affiliation 1:
Immunology and Diabetes Unit, St. Vincent’s Institute, Melbourne, Australia
Affiliation 2:
Department of Medicine, The University of Melbourne, Melbourne, Australia
For correspondence:
hthomas@svi.edu.au
Bio-protocol author page:
a3229
,
date: 6/20/2016, 2673 views,
0 Q&A
DOI:
https://doi.org/10.21769/BioProtoc.1840
.
[Abstract] The islets of Langerhans are clusters of endocrine cells located within the pancreas. Insulin-producing beta cells are the major cell type within islets, with glucagon-producing alpha cells and somatostatin-producing delta cells the other major cell types. The beta cells are the target of immune-mediated destruction in type 1 diabetes (Graham
et al.
, ...
Preparation of Single Cell Suspensions from Mouse Aorta
Authors:
Desheng Hu
Desheng Hu
Affiliation 1:
Institute for Immunology, Ludwig-Maximilians-University Munich, Munich, Germany
Affiliation 2:
Institute of Molecular Immunology, Helmholtz Zentrum Munich, Munich, Germany
For correspondence:
desheng.hu@med.uni-muenchen.de
Bio-protocol author page:
a3210
,
Changjun Yin
Changjun Yin
Affiliation:
Institute for Cardiovascular Prevention, Ludwig-Maximilians-University Munich, Munich, Germany
Bio-protocol author page:
a3208
,
Sarajo Mohanta
Sarajo Mohanta
Affiliation:
Institute for Cardiovascular Prevention, Ludwig-Maximilians-University Munich, Munich, Germany
Bio-protocol author page:
a3207
,
Christian Weber
Christian Weber
Affiliation:
Institute for Cardiovascular Prevention, Ludwig-Maximilians-University Munich, Munich, Germany
Bio-protocol author page:
a3209
and
Andreas J. R. Habenicht
Andreas J. R. Habenicht
Affiliation:
Institute for Cardiovascular Prevention, Ludwig-Maximilians-University Munich, Munich, Germany
Bio-protocol author page:
a3211
,
date: 6/5/2016, 2207 views,
0 Q&A
DOI:
https://doi.org/10.21769/BioProtoc.1832
.
[Abstract] Atherosclerosis is a chronic inflammatory disease of the arterial wall characterized by lipid deposition, plaque formation, and immune cell infiltration. Innate and adaptive immune cells infiltrate the artery during development of the disease. Moreover, advanced disease leads to formation of artery tertiary lymphoid organs in the adventitia (Grabner ...
In vitro
Differentiation of Murine Innate Lymphoid Cells from Common Lymphoid Progenitor Cells
Authors:
Corey Seehus
Corey Seehus
Affiliation:
Research Division of Immunology, Departments of Biomedical Sciences and Medicine, Cedars Sinai Medical Center, Los Angeles, USA
For correspondence:
corey.seehus@csmc.edu
Bio-protocol author page:
a3005
and
Jonathan Kaye
Jonathan Kaye
Affiliation:
Research Division of Immunology, Departments of Biomedical Sciences and Medicine, Cedars Sinai Medical Center, Los Angeles, USA
Bio-protocol author page:
a3006
,
date: 3/20/2016, 1769 views,
0 Q&A
DOI:
https://doi.org/10.21769/BioProtoc.1770
.
[Abstract] Subtypes of innate lymphoid cells (ILC), defined based on their cytokine secretion profiles and transcription factor expression, are important for host protection from pathogens and maintaining tissue homeostasis. ILCs develop from common lymphoid progenitors (CLP) in the bone marrow. Using the methods described here, we have previously shown that ...
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In vitro
Culture of Human PBMCs
Authors:
Santosh K Panda
Santosh K Panda
Affiliation:
Infectious Disease Biology, Institute of Life Sciences, Bhubaneswar, India
Bio-protocol author page:
a221
and
Balachandran Ravindran
Balachandran Ravindran
Affiliation:
Infectious Disease Biology, Institute of Life Sciences, Bhubaneswar, India
For correspondence:
ravindran8@gmail.com
Bio-protocol author page:
a222
,
date: 2/5/2013, 45547 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 Chen
Affiliation:
Department of Genetics, Stanford University, Stanford, USA
For correspondence:
rcchen@jfkbio.com
Bio-protocol author page:
a34
,
date: 2/5/2012, 36280 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 ...
Isolation of Human PBMCs
Authors:
Santosh K Panda
Santosh K Panda
Affiliation:
Infectious Disease Biology, Institute of Life Sciences, Bhubaneswar, India
Bio-protocol author page:
a221
and
Balachandran Ravindran
Balachandran Ravindran
Affiliation:
Infectious Disease Biology, Institute of Life Sciences, Bhubaneswar, India
For correspondence:
ravindran8@gmail.com
Bio-protocol author page:
a222
,
date: 2/5/2013, 23266 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....
Bronchoalveolar Lavage and Lung Tissue Digestion
Authors:
Hongwei Han
Hongwei Han
Affiliation:
Immunology Program, Benaroya Research Institute, Seattle, USA
For correspondence:
hhan@benaroyaresearch.org
Bio-protocol author page:
a544
and
Steven F. Ziegler
Steven F. Ziegler
Affiliation:
Immunology Program, Benaroya Research Institute, Seattle, USA
For correspondence:
sziegler@benaroyaresearch.org
Bio-protocol author page:
a543
,
date: 8/20/2013, 14054 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 Bethunaickan
Affiliation:
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 Davidson
Affiliation:
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, 13616 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 ...
Harvest and Culture of Mouse Peritoneal Macrophages
Authors:
Mingfang Lu
Mingfang Lu
Affiliation:
Internal Medicine, University of Texas Southwestern Medical School, Dallas, TX, USA
Bio-protocol author page:
a990
and
Alan W. Varley
Alan W. Varley
Affiliation:
Internal Medicine, University of Texas Southwestern Medical School, Dallas, TX, USA
For correspondence:
alanwaynevarley@gmail.com
Bio-protocol author page:
a991
,
date: 11/20/2013, 13130 views,
1 Q&A
DOI:
https://doi.org/10.21769/BioProtoc.976
.
[Abstract] Peritoneal macrophages are used as primary macrophages in lots of studies, mainly because they are easy to obtain. Injection of thioglycollate broth i.p. induces inflammatory responses and elicits large numbers of macrophages. This protocol can be used for harvesting resident or thioglycollate-elicited ...
Isolation, Purification, and Culture of Primary Murine Microglia Cells
Authors:
Xuqin Chen
Xuqin Chen
Affiliation:
Department of Neurology, Affiliated Children's Hospital, Soochow University, Suzhou, China
For correspondence:
xuqinlili@yahoo.com
Bio-protocol author page:
a202
,
Ye Zhang
Ye Zhang
Affiliation:
Department of Neurology, Affiliated Children's Hospital, Soochow University, Suzhou, China
Bio-protocol author page:
a203
,
Gaitri Sadadcharam
Gaitri Sadadcharam
Affiliation:
Department of Neurology, Affiliated Children's Hospital, Soochow University, Suzhou, China
Bio-protocol author page:
a204
,
Weili Cui
Weili Cui
Affiliation:
Department of Neurology, Affiliated Children's Hospital, Soochow University, Suzhou, China
Bio-protocol author page:
a205
and
Jiang Huai Wang
Jiang Huai Wang
Affiliation:
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, 12889 views,
0 Q&A
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 ...
Generation of Mouse Bone Marrow-Derived Dendritic Cells (BM-DCs)
Authors:
Francesca Granucci
Francesca Granucci
Affiliation:
Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milan, Italy
For correspondence:
francesca.granucci@unimib.it
Bio-protocol author page:
a59
,
Renato Ostuni
Renato Ostuni
Affiliation:
Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milan, Italy
Bio-protocol author page:
a58
and
Ivan Zanoni
Ivan Zanoni
Affiliation:
Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milan, Italy
For correspondence:
ivan.zanoni@unimib.it
Bio-protocol author page:
a54
,
date: 6/20/2012, 10004 views,
0 Q&A
DOI:
https://doi.org/10.21769/BioProtoc.226
.
[Abstract] Generating mouse dendritic cells from bone-marrow progenitor cells is a useful tool to study biological functions of mouse dendritic cells. Dendritic cells are one of the major populations of phagocytes able to activate both innate and adaptive immune cells. ...
Cell Isolation of Spleen Mononuclear Cells
Author:
Benno Weigmann
Benno Weigmann
Affiliation:
Department of Medicine 1, University Clinic, Erlangen, Germany
For correspondence:
benno.weigmann@uk-erlangen.de
Bio-protocol author page:
a321
,
date: 5/5/2013, 9719 views,
0 Q&A
DOI:
https://doi.org/10.21769/BioProtoc.689
.
[Abstract] This method allows you to isolate different subclass mononuclear cells, like B-cells, T cells, CD4+ and CD8+ T, from mouse spleen. By conjugating cells with specific antibodies and subsequently magnetic beads isolation, using the technique from Miltenyi, this allows a high purity....
Generation of Human iNKT Cell Lines
Authors:
Xiangming Li
Xiangming Li
Affiliation:
HIV and Malaria Vaccine Program, Aaron Diamond AIDS Research Center, New York, USA
Bio-protocol author page:
a440
,
Moriya Tsuji
Moriya Tsuji
Affiliation:
HIV and Malaria Vaccine Program, Aaron Diamond AIDS Research Center, New York, USA
Bio-protocol author page:
a441
,
Jonathan Schneck
Jonathan Schneck
Affiliation:
Department of Pathology, Johns Hopkins University, Baltimore, USA
Bio-protocol author page:
a443
and
Tonya J. Webb
Tonya J. Webb
Affiliation:
Microbiology and Immunology Department, University of Maryland School of Medicine, Baltimore, USA
For correspondence:
twebb@som.umaryland.edu
Bio-protocol author page:
a315
,
date: 3/20/2013, 9404 views,
3 Q&A
DOI:
https://doi.org/10.21769/BioProtoc.418
.
[Abstract] Natural killer T (NKT) cells comprise an important immunoregulatory T cell subset and express cell surface proteins characteristic of both natural killer cells and T cells. Invariant NKT (iNKT) cells are activated by lipid antigen presented in the context of CD1d molecules, in contrast to classic T ...
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