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Adhesion Assay for Murine Bone Marrow Hematopoietic Stem Cells

Featured protocol,  Authors: Seymen Avci
Seymen AvciAffiliation: Weizmann Institute of Science, Immunology department, Rehovot, Israel
Bio-protocol author page: a4075
Shiri Gur-Cohen
Shiri Gur-CohenAffiliation: Weizmann Institute of Science, Immunology department, Rehovot, Israel
Bio-protocol author page: a4060
Francesca Avemaria
Francesca AvemariaAffiliation: Weizmann Institute of Science, Immunology department, Rehovot, Israel
Bio-protocol author page: a4059
 and Tsvee Lapidot
Tsvee LapidotAffiliation: Weizmann Institute of Science, Immunology department, Rehovot, Israel
For correspondence: Tsvee.Lapidot@weizmann.ac.il
Bio-protocol author page: a4121
date: 2/20/2017, 39 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2135.

Brief version appeared in Nat Med, Nov 2015
Hematopoietic stem cells (HSCs) are defined by their functional abilities to self-renew and to give rise to all mature blood and immune cell types throughout life. Most HSCs are retained in a non-motile quiescent state within a specialized protective microenvironment in the bone marrow (BM) termed the niche. HSCs are typically distinguished from other adult stem cells by their motility capacity. Movement of HSCs across the physical barrier of the marrow extracellular matrix and blood vessel endothelial cells is facilitated by suppression of adhesion interactions, which are essential to preserve the stem cells retained within their BM niches. Importantly, homing of HSCs to the BM following clinical transplantation is a crucial first step for the repopulation of ablated BM as in the case of curative treatment strategies for hematologic malignancies. The homing process ends with selective access and anchorage of HSCs to their specialized niches within the BM. Adhesion molecules are targets to either enhance homing in cases of stem cell transplantation or reduce BM retention to harvest mobilized HSCs from the blood of matched donors. A major adhesion protein which is functionally expressed on HSCs and is involved in their homing and retention is the integrin alpha4beta1 (Very late antigen-4; VLA4). In this protocol we introduce an adhesion assay optimized for VLA4 expressing murine bone marrow stem cells. This assay quantifies adherent HSCs by flow cytometry with HSC enriching cell surface markers subsequent to the isolation of VLA4 expressing adherent cells.

VLA-4 Affinity Assay for Murine Bone Marrow-derived Hematopoietic Stem Cells

Featured protocol,  Authors: Francesca Avemaria
Francesca AvemariaAffiliation: Department of Immunology, Weizmann Institute of Science, Rehovot, Israel
Bio-protocol author page: a4059
Shiri Gur-Cohen
Shiri Gur-CohenAffiliation: Department of Immunology, Weizmann Institute of Science, Rehovot, Israel
Bio-protocol author page: a4060
Seymen Avci
Seymen AvciAffiliation: Department of Immunology, Weizmann Institute of Science, Rehovot, Israel
Bio-protocol author page: a4075
 and Tsvee Lapidot
Tsvee LapidotAffiliation: Department of Immunology, Weizmann Institute of Science, Rehovot, Israel
For correspondence: Tsvee.Lapidot@weizmann.ac.il
Bio-protocol author page: a4121
date: 2/20/2017, 38 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2134.

Brief version appeared in Nat Med, Nov 2015
Hematopoietic stem cells (HSCs) are defined by their functional ability to self-renew and to differentiate into all blood cell lineages. The majority of HSC reside in specific anatomical locations in the bone marrow (BM) microenvironment, in a quiescent non motile mode. Adhesion interactions between HSCs and their supporting BM microenvironment cells are critical for maintaining stem cell quiescence and protection from DNA damaging agents to prevent hematology failure and death. Multiple signaling proteins play a role in controlling retention and migration of bone marrow HSCs. Adhesion molecules are involved in both processes regulating hematopoiesis and stem- and progenitor-cell BM retention, migration and development. The mechanisms underlying the movement of stem cells from and to the marrow have not been completely elucidated and are still an object of intense study. One important aspect is the modification of expression and affinity of adhesion molecules by stem and progenitor cells which are required both for stem cell retention, migration and development. Adhesion is regulated by expression of the adhesion molecules, their affinity and avidity. Affinity regulation is related to the molecular binding recognition and bond strength. Here, we describe the in vitro FACS assay used in our research to explore the expression, affinity and function of the integrin α4β1 (also termed VLA-4) for murine bone marrow retained EPCR+ long term repopulation HSC (LT-HSC) (Gur-Cohen et al., 2015).

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

Featured protocol,  Authors: Jae Hong Im
Jae Hong ImAffiliation: CRUK-MRC Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK
Bio-protocol author page: a4021
 and Ruth J. Muschel
Ruth J. MuschelAffiliation: CRUK-MRC Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK
For correspondence: ruth.muschel@oncology.ox.ac.uk
Bio-protocol author page: a4022
date: 2/20/2017, 38 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2132.

Brief version appeared in Blood, Mar 2012
Platelets and coagulation have long been known to be essential for metastasis in experimental models. In order to study the interactions between tumor cells, platelets and endothelium, we have adapted methods used in coagulation research for the isolation of platelets and their reintroduction into mice. Anti-coagulated murine blood served as the source for platelets. Platelets were separated from other elements of the whole blood by centrifugation. Here the critical elements are first inhibition of coagulation and second isolation and maintenance of the platelets in the presence of inhibitors of platelet activation. We then used the vital dye PKH26 to fluorescently label the platelets. Infusion of these labelled platelets allows microscopic observation of the introduced platelets. After reintroduction, these platelets appear to function normally and comprise approximately 50% of the total platelets. Because they are fluorescently labelled, they can easily be identified. Finally it would be possible to use these methods for the determination of specific effects of altered gene expression in platelets by using platelets from genetically engineered mice. These methods have facilitated study of the interactions between platelets and tumor cells in tissue culture and in murine models. They would also be applicable to video microscopy. Here we provide details of the methods we have used for platelet isolation from mice and their staining for further microscopy and re-introduction into mice.

A Murine Orthotopic Allograft to Model Prostate Cancer Growth and Metastasis

Featured protocol,  Authors: Robert M. Hughes
Robert M. HughesAffiliation 1: The James Buchanan Brady Urological Institute, Department of Urology, Johns Hopkins School of Medicine, Baltimore, USA
Affiliation 2: The Department of Oncology, Johns Hopkins School of Medicine, Baltimore, USA
Affiliation 3: The Sidney Kimmel Cancer Center, Johns Hopkins School of Medicine, Baltimore, USA
Bio-protocol author page: a4138
Brian W. Simons
Brian W. SimonsAffiliation: The James Buchanan Brady Urological Institute, Department of Urology, Johns Hopkins School of Medicine, Baltimore, USA
Bio-protocol author page: a4139
 and Paula J. Hurley
Paula J. HurleyAffiliation 1: The James Buchanan Brady Urological Institute, Department of Urology, Johns Hopkins School of Medicine, Baltimore, USA
Affiliation 2: The Department of Oncology, Johns Hopkins School of Medicine, Baltimore, USA
Affiliation 3: The Sidney Kimmel Cancer Center, Johns Hopkins School of Medicine, Baltimore, USA
For correspondence: phurley2@jhmi.edu
Bio-protocol author page: a4140
date: 2/20/2017, 32 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2137.

Brief version appeared in Cancer Res, Oct 2015
Prostate cancer is one of the most common cancers in men in the United States. Comprehensive understanding of the biology contributing to prostate cancer will have important clinical implications. Animal models have greatly impacted our knowledge of disease and will continue to be a valuable resource for future studies. Herein, we describe a detailed protocol for the orthotopic engraftment of a murine prostate cancer cell line (Myc-CaP) into the anterior prostate of an immune competent mouse.

Quantitative Analysis of Exosome Secretion Rates of Single Cells

Featured protocol,  Authors: Yu-Jui Chiu*
Yu-Jui ChiuAffiliation: Materials Science and Engineering Program, University of California at San Diego
La Jolla, California, USA
For correspondence: fenixroger@gmail.com
Bio-protocol author page: a4115
Wei Cai*
Wei CaiAffiliation: Materials Science and Engineering Program, University of California at San Diego
La Jolla, California, USA
Bio-protocol author page: a4111
Tiffany Lee
Tiffany LeeAffiliation: Radiological Health Engineering Program, Texas A & M University, Texas, USA
Bio-protocol author page: a4112
Julia Kraimer
Julia KraimerAffiliation: Biomedical Engineering, University of Cincinnati, Cincinnati, Ohio, USA
Bio-protocol author page: a4113
 and Yu-Hwa Lo
Yu-Hwa LoAffiliation 1: Materials Science and Engineering Program, University of California at San Diego
La Jolla, California, USA
Affiliation 2: Department of Electrical and Computer Engineering University of California at San Diego
La Jolla, California, USA
Bio-protocol author page: a4125
 (*contributed equally to this work) date: 2/20/2017, 33 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2143.

Brief version appeared in Small, Jul 2016
To study the inhomogeneity within a cell population including exosomes properties such as exosome secretion rate of cells and surface markers carried by exosomes, we need to quantify and characterize those exosomes secreted by each individual cell. Here we develop a method to collect and analyze exosomes secreted by an array of single cells using antibody-modified glass slides that are position-registered to each single cell. After each collection, anti-body conjugated quantum dots are used to label exosomes to allow counting and analysis of exosome surface proteins. Detailed studies of exosome properties related to cell behaviors such as responses to drugs and stress at single cell resolution can be found in the publication (Chiu et al., 2016).

In situ Hybridization (ISH) and Quantum Dots (QD) of miRNAs

Featured protocol,  Authors: Sajni Josson
Sajni JossonAffiliation: Uro-Oncology Research Program, Department of Medicine, Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, USA
Present address: Neostrata Inc, Princeton, USA
For correspondence: sajnij@gmail.com
Bio-protocol author page: a4072
Murali Gururajan
Murali GururajanAffiliation: Uro-Oncology Research Program, Department of Medicine, Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, USA
Present address: Bristol-Myers Squibb Inc, Princeton, USA
For correspondence: gururajanmurali@gmail.com
Bio-protocol author page: a4073
 and Leland W.K. Chung
Leland W.K. ChungAffiliation: Uro-Oncology Research Program, Department of Medicine, Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, USA
For correspondence: leland.chung@cshs.org
Bio-protocol author page: a4074
date: 2/20/2017, 36 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2138.

Brief version appeared in Oncogene, May 2015
miRNA are short non-coding RNA which inhibit translation of mRNA. miRNA regulate several cellular processes. Certain miRNA are known to induce oncogenesis. miRNA can be measured by real-time PCR and be imaged using a combination of in situ hybridization (ISH) and quantum dots (QD). The advantage of using quantum dots is that several miRNA can be simultaneously measured using multiplexed QD. Additionally, miRNA can be visualized in different regions of the tissue. Since miRNA are biomarkers of various disease states, miRNA can be visualized and quantitated in tissue sections for diagnostic and prognostic purposes. Here we describe ISH-QD analysis of tissue sections. Tissue sections from xenografts or clinical specimens are used. These are deparaffinized, treated with Proteinase K and hybridized with a biotin-probe to specific to the miRNA. The in situ hybridization is performed by labeling the biotin-probes and followed by labeling with streptavidin tagged quantum dots. Image acquisition of the quantum dots is performed and analyzed for the miRNA expression levels. Combining ISH and QD gives a powerful tool to detect miRNA in different cells of the tissue.

miRNA Characterization from the Extracellular Vesicles

Featured protocol,  Authors: Sajni Josson
Sajni JossonAffiliation 1: Uro-Oncology Research Program, Department of Medicine, Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, USA
Affiliation 2: Neostrata Inc, Princeton, USA
For correspondence: gururajanmurali@gmail.com
Bio-protocol author page: a4072
Murali Gururajan
Murali GururajanAffiliation 1: Uro-Oncology Research Program, Department of Medicine, Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, USA
Affiliation 2: Bristol-Myers Squibb Inc, Princeton, USA
For correspondence: gururajanmurali@gmail.com
Bio-protocol author page: a4073
 and Leland W.K. Chung
Leland W.K. ChungAffiliation: Uro-Oncology Research Program, Department of Medicine, Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, USA
For correspondence: leland.chung@cshs.org
Bio-protocol author page: a4074
date: 2/20/2017, 33 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2139.

Brief version appeared in Oncogene, May 2015
Cancer cells and cancer associated stromal cells co-evolve secrete extracelluar vesicles to the surrounding regions and regulate several processes involved in cancer metastasis. miRNAs have been known to be mediators of cancer progression and metastasis. miRNAs consist of short noncoding RNA. miRNAs are stable in extracellular fluids such as serum, plasma and urine. miRNAs are secreted by cells in normal and diseased conditions. miRNAs signatures have been identified specific to certain disease conditions. Therefore they are valuable biomarkers for different diseases. In our study we identified certain miRNAs, miR-409-3p and miR-409-5p, which were secreted by activated stromal fibroblast cells and were taken up by cancer cells to induce explosive tumor growth, through activation of epithelial to mesenchymal transition of cancer cells. Here we describe a procedure to determine miRNAs (miR-409-3p and miR-409-5p) in extracellular vesicles, which were secreted by prostate cancer stromal cells expressing miR-409. In this procedure, conditioned media from the stromal fibroblasts was used to extract the vesicular fraction. RNA was purified from the vesicular fraction, and specific miRNA was reverse transcribed and quantitated using real-time PCR assay.

Analysis of Cancer Stromal Reaction Using an O-ring Co-culture Assay

Featured protocol,  Author: Vivien Jane Coulson-Thomas
Vivien Jane Coulson-ThomasAffiliation: University of Houston, College of Optometry, the Ocular Surface Institute (TOSI), Houston, TX, USA
For correspondence: vcoulsonthomas@gmail.com
Bio-protocol author page: a1653
date: 2/20/2017, 60 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2131.

Brief version appeared in Exp Cell Res, Nov 2010
We have developed a 2D heterotypic co-culture technique between fibroblasts and cancer cells that enables the study of the stromal reaction. For such, stromal cells are seeded and cultured immediately around a tumour cell line, and the cells establish cell-cell contacts, as well as a gradient of soluble factors throughout the stromal cells, similar to that found in tissues. Thus, this system also enables the researcher to distinguish between events that are caused by direct cell-cell contact and secreted factors.

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

Featured protocol,  Authors: Anna Waldhuber
Anna WaldhuberAffiliation: Molekulare Pädiatrie, Dr. von Haunersches Kinderspital, Ludwig-Maximilians-Universität, München, Germany
Bio-protocol author page: a4062
Manoj Puthia
Manoj PuthiaAffiliation: Department of Microbiology, Immunology and Glycobiology (MIG), Institute of Laboratory Medicine, Lund University, Lund, Sweden
Bio-protocol author page: a4063
Andreas Wieser
Andreas WieserAffiliation: Max von Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie, Ludwig-Maximilians-Universität, München, Germany
Bio-protocol author page: a4064
Catharina Svanborg
Catharina SvanborgAffiliation: Department of Microbiology, Immunology and Glycobiology (MIG), Institute of Laboratory Medicine, Lund University, Lund, Sweden
Bio-protocol author page: a4065
 and Thomas Miethke
Thomas Miethke Affiliation: Institute of Medical Microbiology and Hygiene, Medical Faculty of Mannheim, University of Heidelberg, Mannheim, Germany
For correspondence: thomas.miethke@medma.uni-heidelberg.de
Bio-protocol author page: a4056
date: 2/5/2017, 139 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2129.

Brief version appeared in J Clin Invest, Jul 2016
This urinary tract infection model was used to monitor the efficacy of a new virulence factor of the uropathogenic Escherichia coli strain CFT073 in vivo. The new virulence factor which we designated TIR-containing protein C (TcpC) blocks Toll-like receptor signaling and the NLRP3 inflammasome signaling cascade by interacting with key components of both pattern recognition receptor systems (Cirl et al., 2008; Waldhuber et al., 2016). We infected wild type and knock-out mice with wildtype CFT073 and a mutant CFT073 strain lacking tcpC. This protocol describes how the mice were infected, how CFT073 was prepared and how the infection was monitored. The protocol was derived from our previously published work and allowed us to demonstrate that TcpC is a powerful virulence factor by increasing the bacterial burden of CFT073 in the urine and kidneys. Moreover, TcpC was responsible for the development of kidney abscesses since infection of mice with wildtype but not tcpC-deficient CFT073 mutants caused this complication.

Bioinformatic Analysis for Profiling Drug-induced Chromatin Modification Landscapes in Mouse Brain Using ChlP-seq Data

Featured protocol,  Authors: Yong-Hwee Eddie Loh
Yong-Hwee Eddie LohAffiliation: Fishberg Department of Neuroscience and Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, USA
Bio-protocol author page: a4094
Jian Feng
Jian FengAffiliation: Fishberg Department of Neuroscience and Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, USA
Bio-protocol author page: a4095
Eric Nestler
Eric Nestler Affiliation: Fishberg Department of Neuroscience and Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, USA
Bio-protocol author page: a4096
 and Li Shen
Li ShenAffiliation: Fishberg Department of Neuroscience and Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, USA
For correspondence: li.shen@mssm.edu
Bio-protocol author page: a4097
date: 2/5/2017, 95 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2123.

Brief version appeared in Genome Biol, Apr 2015
Chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-seq) is a powerful technology to profile genome-wide chromatin modification patterns and is increasingly being used to study the molecular mechanisms of brain diseases such as drug addiction. This protocol discusses the typical procedures involved in ChIP-seq data generation, bioinformatic analysis, and interpretation of results, using a chronic cocaine treatment study as a template. We describe an experimental design that induces significant chromatin modifications in mouse brain, and the use of ChIP-seq to derive novel information about the chromatin regulatory mechanisms involved. We describe the bioinformatic methods used to preprocess the sequencing data, generate global enrichment profiles for specific histone modifications, identify enriched genomic loci, find differential modification sites, and perform functional analyses. These ChIP-seq analyses provide many details into the chromatin changes that are induced in brain by chronic exposure to cocaine, and generates an invaluable source of information to understand the molecular mechanisms underlying drug addiction. Our protocol provides a standardized procedure for data analysis and can serve as a starting point for any other ChIP-seq projects.

Polysome Fractionation to Analyze mRNA Distribution Profiles

Featured protocol,  Authors: Amaresh C. Panda
Amaresh C. PandaAffiliation: Laboratory of Genetics and Genomics, National Institute on Aging, National Institutes of Health, Biomedical Research Center, Baltimore, USA
For correspondence: amarchpanda@gmail.com
Bio-protocol author page: a3875
Jennifer L. Martindale
Jennifer L. Martindale Affiliation: Laboratory of Genetics and Genomics, National Institute on Aging, National Institutes of Health, Biomedical Research Center, Baltimore, USA
Bio-protocol author page: a3880
 and Myriam Gorospe
Myriam GorospeAffiliation: Laboratory of Genetics and Genomics, National Institute on Aging, National Institutes of Health, Biomedical Research Center, Baltimore, USA
Bio-protocol author page: a3881
date: 2/5/2017, 131 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2126.

Brief version appeared in Nucleic Acids Res, Mar 2016
Eukaryotic cells adapt to changes in external or internal signals by precisely modulating the expression of specific gene products. The expression of protein-coding genes is controlled at the transcriptional and post-transcriptional levels. Among the latter steps, the regulation of translation is particularly important in cellular processes that require rapid changes in protein expression patterns. The translational efficiency of mRNAs is altered by RNA-binding proteins (RBPs) and noncoding (nc)RNAs such as microRNAs (Panda et al., 2014a and 2014b; Abdelmohsen et al., 2014). The impact of factors that regulate selective mRNA translation is a critical question in RNA biology. Polyribosome (polysome) fractionation analysis is a powerful method to assess the association of ribosomes with a given mRNA. It provides valuable information about the translational status of that mRNA, depending on the number of ribosomes with which they are associated, and identifies mRNAs that are not translated (Panda et al., 2016). mRNAs associated with many ribosomes form large polysomes that are predicted to be actively translated, while mRNAs associated with few or no ribosomes are expected to be translated poorly if at all. In sum, polysome fractionation analysis allows the direct determination of translation efficiencies at the level of the whole transcriptome as well as individual mRNAs.

In vitro Assessment of RNA Polymerase I Activity

Featured protocol,  Author: Marzia Govoni
Marzia GovoniAffiliation: Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Bologna, Italy
For correspondence: marzia.govoni@unibo.it
Bio-protocol author page: a4032
date: 2/5/2017, 149 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2120.

Brief version appeared in Oncogene, Feb 2016
In eukaryotic cells transcriptional processes are carried out by three different RNA polymerases: RNA polymerase I which specifically transcribes ribosomal RNA (rRNA), RNA polymerase II which transcribes protein-coding genes to yield messenger RNAs (mRNAs) and small RNAs, while RNA polymerase III transcribes the genes for transfer RNAs and for the smallest species of ribosomal RNA (5S rRNA). This protocol describes an in vitro assay to evaluate the rRNA transcriptional activity of RNA polymerase I. The method measures the quantity of radiolabelled uridine 5’ triphosphate incorporated in ex novo synthesized rRNA molecules by RNA polymerase I, in optimal conditions for the enzyme activity and in the presence of a toxin, α-amanitin, which inhibits RNA polymerase II and III without affecting RNA polymerase I (Novello and Stirpe, 1970).

Cued Rat Gambling Task

Featured protocol,  Authors: Michael M. Barrus
Michael M. BarrusAffiliation: Department of Psychology, University of British Columbia, Vancouver, British Columbia, Canada
Bio-protocol author page: a4033
 and Catharine A. Winstanley
Catharine A. WinstanleyAffiliation: Department of Psychology, University of British Columbia, Vancouver, British Columbia, Canada
For correspondence: cwinstanley@psych.ubc.ca
Bio-protocol author page: a4034
date: 2/5/2017, 112 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2118.

Brief version appeared in J Neurosci, Jan 2016
The ability of salient cues to serve as powerful motivators has long been recognized in models of drug addiction, but little has been done to investigate their effects on complex decision making. The Cued rat Gambling Task (CrGT) is an operant behavioural task which pairs salient, audiovisual cues with the delivery of sucrose pellet rewards on complex schedules of reinforcement that involve both sugar pellet ‘wins’ and timeout penalty ‘losses’. The task was designed with the intention of providing insight into the influence of such cues on decision making in a manner that models human gambling.

Heterochronic Pellet Assay to Test Cell-cell Communication in the Mouse Retina

Featured protocol,  Authors: Nobuhiko Tachibana
Nobuhiko TachibanaAffiliation 1: Biological Sciences Platform, Sunnybrook Research Institute, University of Toronto, Toronto, Canada
Affiliation 2: Department of Biochemistry and Molecular Biology, Alberta Children’s Hospital Research Institute and Hotchkiss Brain Institute, University of Calgary, Calgary, Canada
Bio-protocol author page: a4105
Dawn Zinyk
Dawn ZinykAffiliation 1: Biological Sciences Platform, Sunnybrook Research Institute, University of Toronto, Toronto, Canada
Affiliation 2: Department of Biochemistry and Molecular Biology, Alberta Children’s Hospital Research Institute and Hotchkiss Brain Institute, University of Calgary, Calgary, Canada
Bio-protocol author page: a4106
Randy Ringuette
Randy RinguetteAffiliation: Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Canada
Bio-protocol author page: a4107
Valerie Wallace
Valerie WallaceAffiliation 1: Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Canada
Affiliation 2: Donald K. Johnson Eye Institute, Krembil Research Institute, University Health Network, Toronto, Canada
Affiliation 3: Department of Ophthalmology and Vision Sciences and Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada
For correspondence: vwallace@uhnresearch.ca
Bio-protocol author page: a4108
 and Carol Schuurmans
Carol SchuurmansAffiliation 1: Biological Sciences Platform, Sunnybrook Research Institute, University of Toronto, Toronto, Canada
Affiliation 2: Department of Biochemistry and Molecular Biology, Alberta Children’s Hospital Research Institute and Hotchkiss Brain Institute, University of Calgary, Calgary, Canada
For correspondence: cschuurm@sri.utoronto.ca
Bio-protocol author page: a4109
date: 2/5/2017, 131 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2117.

Brief version appeared in J Neurosci, Sep 2016
All seven retinal cell types that make up the mature retina are generated from a common, multipotent pool of retinal progenitor cells (RPCs) (Wallace, 2011). One way that RPCs know when sufficient numbers of particular cell-types have been generated is through negative feedback signals, which are emitted by differentiated cells and must reach threshold levels to block additional differentiation of that cell type. A key assay to assess whether negative feedback signals are emitted by differentiated cells is a heterochronic pellet assay in which early stage RPCs are dissociated and labeled with BrdU, then mixed with a 20-fold excess of dissociated differentiated cells. The combined cells are then re-aggregated and cultured as a pellet on a membrane for 7-10 days in vitro. During this time frame, RPCs will differentiate, and the fate of the BrdU+ RPCs can be assessed using cell type-specific markers. Investigators who developed this pellet assay initially demonstrated that neonatal RPCs give rise to rods on an accelerated schedule compared to embryonic RPCs when the two cell types are mixed together (Watanabe and Raff, 1990; Watanabe et al., 1997). We have used this assay to demonstrate that sonic hedgehog (Shh), which we found acts as a negative regulator of retinal ganglion cell (RGC) differentiation, promotes RPC proliferation (Jensen and Wallace, 1997; Ringuette et al., 2014). More recently we modified the heterochronic pellet assay to assess the role of feedback signals for retinal amacrine cells, identifying transforming growth factor β2 (Tgfβ2) as a negative feedback signal, and Pten as a modulator of the Tgfβ2 response (Ma et al., 2007; Tachibana et al., 2016). This assay can be adapted to other lineages and tissues to assess cell-cell interactions between two different cell-types (heterotypic) in either an isochronic or heterochronic manner.

A Streamlined Method for the Preparation of Growth Factor-enriched Thermosensitive Hydrogels from Soft Tissue

Featured protocol,  Authors: Christopher J. Poon
Christopher J. PoonAffiliation: O’Brien Institute Department of SVI, St. Vincent’s Institute of Medical Research, Melbourne, Australia
For correspondence: christopher.poon@alumni.unimelb.edu.au
Bio-protocol author page: a4044
Shaun S. Tan
Shaun S. TanAffiliation: O’Brien Institute Department of SVI, St. Vincent’s Institute of Medical Research, Melbourne, Australia
Bio-protocol author page: a4045
Sholeh W. Boodhun
Sholeh W. BoodhunAffiliation: O’Brien Institute Department of SVI, St. Vincent’s Institute of Medical Research, Melbourne, Australia
Bio-protocol author page: a4046
Keren M. Abberton
Keren M. Abberton Affiliation: O’Brien Institute Department of SVI, St. Vincent’s Institute of Medical Research, Melbourne, Australia
Bio-protocol author page: a4047
 and Wayne A. Morrison
Wayne A. MorrisonAffiliation: O’Brien Institute Department of SVI, St. Vincent’s Institute of Medical Research, Melbourne, Australia
Bio-protocol author page: a4048
date: 2/5/2017, 114 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2128.

Brief version appeared in Acta Biomater, Mar 2013
Hydrogels are an ideal medium for the expansion of cells in three dimensions. The ability to induce cell expansion and differentiation in a controlled manner is a key goal in tissue engineering. Here we describe a detailed method for producing hydrogels from soft tissues with an emphasis on adipose tissue. In this method, soluble, extractable proteins are recovered from the tissue and stored while the remaining insoluble tissue is processed and solubilised. Once the tissue has been sufficiently solubilised, the extracted proteins are added. The resulting product is a thermosensitive hydrogel with proteins representative of the native tissue. This method addresses common issues encountered when working with some biomaterials, such as high lipid content, DNA contamination, and finding an appropriate sterilisation method. Although the focus of this article is on adipose tissue, using this method we have produced hydrogels from other soft tissues including muscle, liver, and cardiac tissue.

Adhesion Assay for Murine Bone Marrow Hematopoietic Stem Cells

Authors: Seymen Avci
Seymen AvciAffiliation: Weizmann Institute of Science, Immunology department, Rehovot, Israel
Bio-protocol author page: a4075
Shiri Gur-Cohen
Shiri Gur-CohenAffiliation: Weizmann Institute of Science, Immunology department, Rehovot, Israel
Bio-protocol author page: a4060
Francesca Avemaria
Francesca AvemariaAffiliation: Weizmann Institute of Science, Immunology department, Rehovot, Israel
Bio-protocol author page: a4059
 and Tsvee Lapidot
Tsvee LapidotAffiliation: Weizmann Institute of Science, Immunology department, Rehovot, Israel
For correspondence: Tsvee.Lapidot@weizmann.ac.il
Bio-protocol author page: a4121
date: 2/20/2017, 39 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2135.

[Abstract] Hematopoietic stem cells (HSCs) are defined by their functional abilities to self-renew and to give rise to all mature blood and immune cell types throughout life. Most HSCs are retained in a non-motile quiescent state within a specialized protective microenvironment in the bone marrow (BM) termed the ...

VLA-4 Affinity Assay for Murine Bone Marrow-derived Hematopoietic Stem Cells

Authors: Francesca Avemaria
Francesca AvemariaAffiliation: Department of Immunology, Weizmann Institute of Science, Rehovot, Israel
Bio-protocol author page: a4059
Shiri Gur-Cohen
Shiri Gur-CohenAffiliation: Department of Immunology, Weizmann Institute of Science, Rehovot, Israel
Bio-protocol author page: a4060
Seymen Avci
Seymen AvciAffiliation: Department of Immunology, Weizmann Institute of Science, Rehovot, Israel
Bio-protocol author page: a4075
 and Tsvee Lapidot
Tsvee LapidotAffiliation: Department of Immunology, Weizmann Institute of Science, Rehovot, Israel
For correspondence: Tsvee.Lapidot@weizmann.ac.il
Bio-protocol author page: a4121
date: 2/20/2017, 38 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2134.

[Abstract] Hematopoietic stem cells (HSCs) are defined by their functional ability to self-renew and to differentiate into all blood cell lineages. The majority of HSC reside in specific anatomical locations in the bone marrow (BM) microenvironment, in a quiescent non motile mode. Adhesion interactions between ...

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

Authors: Jae Hong Im
Jae Hong ImAffiliation: CRUK-MRC Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK
Bio-protocol author page: a4021
 and Ruth J. Muschel
Ruth J. MuschelAffiliation: CRUK-MRC Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK
For correspondence: ruth.muschel@oncology.ox.ac.uk
Bio-protocol author page: a4022
date: 2/20/2017, 38 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2132.

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

A Murine Orthotopic Allograft to Model Prostate Cancer Growth and Metastasis

Authors: Robert M. Hughes
Robert M. HughesAffiliation 1: The James Buchanan Brady Urological Institute, Department of Urology, Johns Hopkins School of Medicine, Baltimore, USA
Affiliation 2: The Department of Oncology, Johns Hopkins School of Medicine, Baltimore, USA
Affiliation 3: The Sidney Kimmel Cancer Center, Johns Hopkins School of Medicine, Baltimore, USA
Bio-protocol author page: a4138
Brian W. Simons
Brian W. SimonsAffiliation: The James Buchanan Brady Urological Institute, Department of Urology, Johns Hopkins School of Medicine, Baltimore, USA
Bio-protocol author page: a4139
 and Paula J. Hurley
Paula J. HurleyAffiliation 1: The James Buchanan Brady Urological Institute, Department of Urology, Johns Hopkins School of Medicine, Baltimore, USA
Affiliation 2: The Department of Oncology, Johns Hopkins School of Medicine, Baltimore, USA
Affiliation 3: The Sidney Kimmel Cancer Center, Johns Hopkins School of Medicine, Baltimore, USA
For correspondence: phurley2@jhmi.edu
Bio-protocol author page: a4140
date: 2/20/2017, 32 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2137.

[Abstract] Prostate cancer is one of the most common cancers in men in the United States. Comprehensive understanding of the biology contributing to prostate cancer will have important clinical implications. Animal models have greatly impacted our knowledge of disease and will continue to be a valuable resource ...

Quantitative Analysis of Exosome Secretion Rates of Single Cells

Authors: Yu-Jui Chiu*
Yu-Jui ChiuAffiliation: Materials Science and Engineering Program, University of California at San Diego
La Jolla, California, USA
For correspondence: fenixroger@gmail.com
Bio-protocol author page: a4115
Wei Cai*
Wei CaiAffiliation: Materials Science and Engineering Program, University of California at San Diego
La Jolla, California, USA
Bio-protocol author page: a4111
Tiffany Lee
Tiffany LeeAffiliation: Radiological Health Engineering Program, Texas A & M University, Texas, USA
Bio-protocol author page: a4112
Julia Kraimer
Julia KraimerAffiliation: Biomedical Engineering, University of Cincinnati, Cincinnati, Ohio, USA
Bio-protocol author page: a4113
 and Yu-Hwa Lo
Yu-Hwa LoAffiliation 1: Materials Science and Engineering Program, University of California at San Diego
La Jolla, California, USA
Affiliation 2: Department of Electrical and Computer Engineering University of California at San Diego
La Jolla, California, USA
Bio-protocol author page: a4125
 (*contributed equally to this work) date: 2/20/2017, 33 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2143.

[Abstract] To study the inhomogeneity within a cell population including exosomes properties such as exosome secretion rate of cells and surface markers carried by exosomes, we need to quantify and characterize those exosomes secreted by each individual cell. Here we develop a method to collect and analyze exosomes ...

In situ Hybridization (ISH) and Quantum Dots (QD) of miRNAs

Authors: Sajni Josson
Sajni JossonAffiliation: Uro-Oncology Research Program, Department of Medicine, Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, USA
Present address: Neostrata Inc, Princeton, USA
For correspondence: sajnij@gmail.com
Bio-protocol author page: a4072
Murali Gururajan
Murali GururajanAffiliation: Uro-Oncology Research Program, Department of Medicine, Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, USA
Present address: Bristol-Myers Squibb Inc, Princeton, USA
For correspondence: gururajanmurali@gmail.com
Bio-protocol author page: a4073
 and Leland W.K. Chung
Leland W.K. ChungAffiliation: Uro-Oncology Research Program, Department of Medicine, Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, USA
For correspondence: leland.chung@cshs.org
Bio-protocol author page: a4074
date: 2/20/2017, 36 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2138.

[Abstract] miRNA are short non-coding RNA which inhibit translation of mRNA. miRNA regulate several cellular processes. Certain miRNA are known to induce oncogenesis. miRNA can be measured by real-time PCR and be imaged using a combination of in situ hybridization (ISH) and quantum dots (QD). The advantage of ...

miRNA Characterization from the Extracellular Vesicles

Authors: Sajni Josson
Sajni JossonAffiliation 1: Uro-Oncology Research Program, Department of Medicine, Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, USA
Affiliation 2: Neostrata Inc, Princeton, USA
For correspondence: gururajanmurali@gmail.com
Bio-protocol author page: a4072
Murali Gururajan
Murali GururajanAffiliation 1: Uro-Oncology Research Program, Department of Medicine, Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, USA
Affiliation 2: Bristol-Myers Squibb Inc, Princeton, USA
For correspondence: gururajanmurali@gmail.com
Bio-protocol author page: a4073
 and Leland W.K. Chung
Leland W.K. ChungAffiliation: Uro-Oncology Research Program, Department of Medicine, Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, USA
For correspondence: leland.chung@cshs.org
Bio-protocol author page: a4074
date: 2/20/2017, 33 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2139.

[Abstract] Cancer cells and cancer associated stromal cells co-evolve secrete extracelluar vesicles to the surrounding regions and regulate several processes involved in cancer metastasis. miRNAs have been known to be mediators of cancer progression and metastasis. miRNAs consist of short noncoding RNA. miRNAs ...

Analysis of Cancer Stromal Reaction Using an O-ring Co-culture Assay

Author: Vivien Jane Coulson-Thomas
Vivien Jane Coulson-ThomasAffiliation: University of Houston, College of Optometry, the Ocular Surface Institute (TOSI), Houston, TX, USA
For correspondence: vcoulsonthomas@gmail.com
Bio-protocol author page: a1653
date: 2/20/2017, 60 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2131.

[Abstract] We have developed a 2D heterotypic co-culture technique between fibroblasts and cancer cells that enables the study of the stromal reaction. For such, stromal cells are seeded and cultured immediately around a tumour cell line, and the cells establish cell-cell contacts, as well as a gradient of soluble ...

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

Authors: Anna Waldhuber
Anna WaldhuberAffiliation: Molekulare Pädiatrie, Dr. von Haunersches Kinderspital, Ludwig-Maximilians-Universität, München, Germany
Bio-protocol author page: a4062
Manoj Puthia
Manoj PuthiaAffiliation: Department of Microbiology, Immunology and Glycobiology (MIG), Institute of Laboratory Medicine, Lund University, Lund, Sweden
Bio-protocol author page: a4063
Andreas Wieser
Andreas WieserAffiliation: Max von Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie, Ludwig-Maximilians-Universität, München, Germany
Bio-protocol author page: a4064
Catharina Svanborg
Catharina SvanborgAffiliation: Department of Microbiology, Immunology and Glycobiology (MIG), Institute of Laboratory Medicine, Lund University, Lund, Sweden
Bio-protocol author page: a4065
 and Thomas Miethke
Thomas Miethke Affiliation: Institute of Medical Microbiology and Hygiene, Medical Faculty of Mannheim, University of Heidelberg, Mannheim, Germany
For correspondence: thomas.miethke@medma.uni-heidelberg.de
Bio-protocol author page: a4056
date: 2/5/2017, 139 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2129.

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

Bioinformatic Analysis for Profiling Drug-induced Chromatin Modification Landscapes in Mouse Brain Using ChlP-seq Data

Authors: Yong-Hwee Eddie Loh
Yong-Hwee Eddie LohAffiliation: Fishberg Department of Neuroscience and Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, USA
Bio-protocol author page: a4094
Jian Feng
Jian FengAffiliation: Fishberg Department of Neuroscience and Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, USA
Bio-protocol author page: a4095
Eric Nestler
Eric Nestler Affiliation: Fishberg Department of Neuroscience and Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, USA
Bio-protocol author page: a4096
 and Li Shen
Li ShenAffiliation: Fishberg Department of Neuroscience and Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, USA
For correspondence: li.shen@mssm.edu
Bio-protocol author page: a4097
date: 2/5/2017, 95 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2123.

[Abstract] Chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-seq) is a powerful technology to profile genome-wide chromatin modification patterns and is increasingly being used to study the molecular mechanisms of brain diseases such as drug addiction. This protocol discusses the typical ...
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Scratch Wound Healing Assay

Author: Yanling Chen
Yanling ChenAffiliation: Department of Immunology, The Scripps Research Institute, La Jolla, USA
For correspondence: ylchen@scripps.edu
Bio-protocol author page: a27
date: 3/5/2012, 52697 views, 6 Q&A
DOI: https://doi.org/10.21769/BioProtoc.100.

[Abstract] The scratch wound healing assay has been widely adapted and modified to study the effects of a variety of experimental conditions, for instance, gene knockdown or chemical exposure, on mammalian cell migration and proliferation. In a typical scratch wound healing assay, a “wound gap” in a cell monolayer ...

Transwell Cell Migration Assay Using Human Breast Epithelial Cancer Cell

Author: Yanling Chen
Yanling ChenAffiliation: Department of Immunology, The Scripps Research Institute, La Jolla, USA
For correspondence: ylchen@scripps.edu
Bio-protocol author page: a27
date: 2/20/2012, 42139 views, 7 Q&A
DOI: https://doi.org/10.21769/BioProtoc.99.

[Abstract] Transwell migration assays have been widely used for studying the motility of different types of cells including metastatic cancer cells. The assay is also useful in screens for compounds that act as chemoattractants or inhibitors of chemotaxis for cells. The assay employs a permeable layer of support, ...

Detection of Intracellular Reactive Oxygen Species (CM-H2DCFDA)

Authors: Rabii Ameziane-El-Hassani
Rabii Ameziane-El-HassaniAffiliation 1: UBRM, Centre National de l'Energie, des Sciences et des Techniques Nucléaires, Rabat, Morocco
Affiliation 2: Institut Gustave Roussy, FRE2939 Centre National de la Recherche Scientifique, Villejuif, France
Bio-protocol author page: a200
 and Corinne Dupuy
Corinne DupuyAffiliation 1: Institut Gustave Roussy, FRE2939 Centre National de la Recherche Scientifique, Villejuif, France
Affiliation 2: University Paris, Sud 11, Orsay, France
For correspondence: dupuy@igr.fr
Bio-protocol author page: a201
date: 1/5/2013, 39443 views, 2 Q&A
DOI: https://doi.org/10.21769/BioProtoc.313.

[Abstract] Reactive oxygen species (ROS) play a critical role in cellular physiopathology. ROS are implicated in cell proliferation, signaling pathways, oxidative defense mechanisms responsible for killing of bacteria, thyroid hormonosynthesis, etc. The cellular Redox homeostasis is balanced by oxidants and antioxidants ...

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, 39147 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 ...

[Bio101] Cell Proliferation Assay by Flow Cytometry (BrdU and PI Staining)

Author: Hui Zhu
Hui ZhuAffiliation: Department of Genetics, Stanford University, Stanford, USA
For correspondence: huizhu@stanford.edu
Bio-protocol author page: a32
date: 4/5/2012, 37767 views, 2 Q&A
DOI: https://doi.org/10.21769/BioProtoc.198.

[Abstract] Cell Proliferation assays include an important set of fluorescence-based tests that can monitor cell health and cell division by evaluating DNA synthesis through thymidine incorporation. Bromodeoxyuridine (5-bromo-2'-deoxyuridine, BrdU) is a synthetic nucleoside that is an analogue of thymidine. BrdU ...

Clonogenic Assay

Author: Xiaodong Yang
Xiaodong YangAffiliation: Department of Neurology, University of California, San Francisco, USA
For correspondence: yangxiaodong1@yahoo.com
Bio-protocol author page: a43
date: 5/20/2012, 35815 views, 5 Q&A
DOI: https://doi.org/10.21769/BioProtoc.187.

[Abstract] Clonogenic assays serve as a useful tool to test whether a given cancer therapy can reduce the clonogenic survival of tumor cells. A colony is defined as a cluster of at least 50 cells that can often only be determined microscopically. A clonogenic assay is the method of choice to determine cell reproductive ...

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, 34381 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 ...

In vitro Human Umbilical Vein Endothelial Cells (HUVEC) Tube-formation Assay

Authors: Josephine MY Ko
Josephine MY KoAffiliation: Clinical Oncology Department, The University of Hong Kong, Hong Kong , Hong Kong SAR
Bio-protocol author page: a100
 and Maria Li Lung
Maria Li LungAffiliation: Clinical Oncology Department, The Univerisity of Hong Kong, Hong Kong, Hong Kong SAR
For correspondence: mlilung@hku.hk
Bio-protocol author page: a101
date: 9/20/2012, 31324 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.260.

[Abstract] Angiogenesis is involved not only in pathological conditions including cancer biology and non-neoplastic diseases, but also many biological processes including reproduction, development and repair. During angiogenesis, endothelial cells (ECs) undergo activation after binding of angiogenic factors to ...

Soft–Agar colony Formation Assay

Author: FengZhi Liu
FengZhi LiuAffiliation: School of Biomedical Sciences, Thomas Jefferson University, Philadelphia, USA
For correspondence: fengzhi6@yahoo.com
Bio-protocol author page: a51
date: 7/5/2012, 30398 views, 6 Q&A
DOI: https://doi.org/10.21769/BioProtoc.220.

[Abstract] Any anchorage–independent growth of tumor cells is estimated by a soft–agar colony formation assay. This protocol provides a general workflow for establishing a soft-agar colony formation assay....

[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, 29764 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 ...
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