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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).

Penetration Assays, Fungal Recovery and Pathogenicity Assays for Verticillium dahliae

Featured protocol,  Authors: Yun-Long Zhao
Yun-Long ZhaoAffiliation: State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China
For correspondence: zyl714@126.com
Bio-protocol author page: a4092
Tao Zhang
Tao Zhang Affiliation: State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China
Bio-protocol author page: a4093
 and Hui-Shan Guo
Hui-Shan GuoAffiliation: State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China
Bio-protocol author page: a2721
date: 2/20/2017, 46 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2133.

Brief version appeared in PLoS Pathog, Jul 2016
Verticillium dahliae is a soil-borne phytopathogenic fungus that infects host roots and proliferates in vascular tissues. The great loss of economically important crop caused by V. dahliae has raised worldwide concern, however, little is known about the mechanism of its pathogenicity (Klosterman et al., 2011; Yadeta and Thomma, 2013). Our recent work has shown that V. dahliae develops hyphopodium as an infection structure to breach plant root cell wall (Zhao et al., 2016). Here, we provide a detailed protocol to analyze the penetration ability and the pathogenicity of V. dahliae as well as recover fungal hyphae from infected cotton stems developed from our previous studies (Zhang et al., 2016a and 2016b; Zhao et al., 2016). Cellophane membrane has been used in inducing appressorium development of foliar pathogens but not root pathogens (Bourett and Howard, 1990). We adopted the method of using the cellophane membrane to induce and assess the development of hyphopodium. Hopefully, it will greatly promote the research of molecular events involved in recognition of the host that regulate infectious development. This protocol is also helpful to identify the key component controlling the pathogenicity of V. dahliae and widen our understanding of the mechanism of plant-microbe interaction.

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.

Automatic Quantification of the Number of Intracellular Compartments in Arabidopsis thaliana Root Cells

Featured protocol,  Authors: Vincent Bayle
Vincent BayleAffiliation: Laboratoire Reproduction et Développement des Plantes, Univ Lyon, ENS de Lyon, Université Claude Bernard Lyon 1, CNRS, INRA, Lyon, France
Bio-protocol author page: a4180
Matthieu Pierre Platre
Matthieu Pierre PlatreAffiliation: Laboratoire Reproduction et Développement des Plantes, Univ Lyon, ENS de Lyon, Université Claude Bernard Lyon 1, CNRS, INRA, Lyon, France
Bio-protocol author page: a4102
 and Yvon Jaillais
Yvon JaillaisAffiliation: Laboratoire Reproduction et Développement des Plantes, Univ Lyon, ENS de Lyon, Université Claude Bernard Lyon 1, CNRS, INRA, Lyon, France
For correspondence: yvon.jaillais@ens-lyon.fr
Bio-protocol author page: a4103
date: 2/20/2017, 36 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2145.

Brief version appeared in Nat Plants, Jun 2016
In the era of quantitative biology, it is increasingly required to quantify confocal microscopy images. If possible, quantification should be performed in an automatic way, in order to avoid bias from the experimenter, to allow the quantification of a large number of samples, and to increase reproducibility between laboratories. In this protocol, we describe procedures for automatic counting of the number of intracellular compartments in Arabidopsis root cells, which can be used for example to study endocytosis or secretory trafficking pathways and to compare membrane organization between different genotypes or treatments. While developed for Arabidopsis roots, this method can be used on other tissues, cell types and plant species.

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.

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.

Synthetic Lethality Screens Using RNAi in Combination with CRISPR-based Knockout in Drosophila Cells

Featured protocol,  Authors: Benjamin E. Housden*
Benjamin E. HousdenAffiliation: Harvard Medical School, Boston, USA
For correspondence: bhousden@genetics.med.harvard.edu
Bio-protocol author page: a4035
Hilary E. Nicholson*
Hilary E. NicholsonAffiliation 1: Harvard Medical School, Boston, USA
Affiliation 2: Dana-Farber Cancer Institute, Boston, USA
Bio-protocol author page: a4036
 and Norbert Perrimon
Norbert PerrimonAffiliation 1: Harvard Medical School, Boston, USA
Affiliation 2: Howard Hughes Medical Institute, Boston, USA
For correspondence: perrimon@receptor.med.harvard.edu
Bio-protocol author page: a4037
 (*contributed equally to this work) date: 2/5/2017, 159 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2119.

Brief version appeared in Sci Signal, Sep 2015
A synthetic lethal interaction is a type of genetic interaction where the disruption of either of two genes individually has little effect but their combined disruption is lethal. Knowledge of synthetic lethal interactions can allow for elucidation of network structure and identification of candidate drug targets for human diseases such as cancer. In Drosophila, combinatorial gene disruption has been achieved previously by combining multiple RNAi reagents. Here we describe a protocol for high-throughput combinatorial gene disruption by combining CRISPR and RNAi. This approach previously resulted in the identification of highly reproducible and conserved synthetic lethal interactions (Housden et al., 2015).

Force Measurement on Mycoplasma mobile Gliding Using Optical Tweezers

Featured protocol,  Authors: Masaki Mizutani
Masaki MizutaniAffiliation: Department of Biology, Graduate School of Science, Osaka City University, Osaka, Japan
Bio-protocol author page: a4091
 and Makoto Miyata
Makoto MiyataAffiliation 1: Department of Biology, Graduate School of Science, Osaka City University, Osaka, Japan
Affiliation 2: The OCU Advanced Research Institute for Natural Science and Technology (OCARINA), Osaka City University, Osaka, Japan
For correspondence: miyata@sci.osaka-cu.ac.jp
Bio-protocol author page: a711
date: 2/5/2017, 134 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2127.

Brief version appeared in mBio, Jun 2016
Dozens of Mycoplasma species, belonging to class Mollicutes form a protrusion at a pole as an organelle. They bind to solid surfaces through the organelle and glide in the direction by a unique mechanism including repeated cycles of bind, pull, and release with sialylated oligosaccharides on host animal cells. The mechanical characters are critical information to understand this unique mechanism involved in their infectious process. In this protocol, we describe a method to measure the force generated by Mycoplasma mobile, the fastest gliding species in Mycoplasma. This protocol should be useful for the studies of many kinds of gliding microorganisms.

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 niche. HSCs are typically distinguished from other ...

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 HSCs and their supporting BM microenvironment cells ...

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. Anti-coagulated murine blood served as the source ...

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 for future studies. Herein, we describe a detailed ...

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 secreted by an array of single cells using antibody-modified ...

Penetration Assays, Fungal Recovery and Pathogenicity Assays for Verticillium dahliae

Authors: Yun-Long Zhao
Yun-Long ZhaoAffiliation: State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China
For correspondence: zyl714@126.com
Bio-protocol author page: a4092
Tao Zhang
Tao Zhang Affiliation: State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China
Bio-protocol author page: a4093
 and Hui-Shan Guo
Hui-Shan GuoAffiliation: State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China
Bio-protocol author page: a2721
date: 2/20/2017, 46 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2133.

[Abstract] Verticillium dahliae is a soil-borne phytopathogenic fungus that infects host roots and proliferates in vascular tissues. The great loss of economically important crop caused by V. dahliae has raised worldwide concern, however, little is known about the mechanism of its pathogenicity (Klosterman et al., 2011; Yadeta and Thomma, 2013). Our recent work ...

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 factors throughout the stromal cells, similar ...

Automatic Quantification of the Number of Intracellular Compartments in Arabidopsis thaliana Root Cells

Authors: Vincent Bayle
Vincent BayleAffiliation: Laboratoire Reproduction et Développement des Plantes, Univ Lyon, ENS de Lyon, Université Claude Bernard Lyon 1, CNRS, INRA, Lyon, France
Bio-protocol author page: a4180
Matthieu Pierre Platre
Matthieu Pierre PlatreAffiliation: Laboratoire Reproduction et Développement des Plantes, Univ Lyon, ENS de Lyon, Université Claude Bernard Lyon 1, CNRS, INRA, Lyon, France
Bio-protocol author page: a4102
 and Yvon Jaillais
Yvon JaillaisAffiliation: Laboratoire Reproduction et Développement des Plantes, Univ Lyon, ENS de Lyon, Université Claude Bernard Lyon 1, CNRS, INRA, Lyon, France
For correspondence: yvon.jaillais@ens-lyon.fr
Bio-protocol author page: a4103
date: 2/20/2017, 36 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2145.

[Abstract] In the era of quantitative biology, it is increasingly required to quantify confocal microscopy images. If possible, quantification should be performed in an automatic way, in order to avoid bias from the experimenter, to allow the quantification of a large number of samples, and to increase reproducibility between laboratories. In this protocol, we ...

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 cascade by interacting with key components of ...

Polysome Fractionation to Analyze mRNA Distribution Profiles

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.

[Abstract] 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 ...
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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] Cell Adhesion 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, 26837 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.98.

[Abstract] Cell adhesion, the binding of a cell to the extracellular matrix (ECM), other cells, or a specific surface, is essential for the growth and survival of the cell and also its communication with other cells. The process of cell adhesion involves a range of biological events such as three-dimensional re-organization ...
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