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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.

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.

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).

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 ...

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 using quantum dots is that several miRNA can be simultaneously ...

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 are stable in extracellular fluids such as serum, ...

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 ...

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 ...

In vitro Assessment of RNA Polymerase I Activity

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.

[Abstract] 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 ...

Assessment of Cellular Redox State Using NAD(P)H Fluorescence Intensity and Lifetime

Authors: Thomas S. Blacker
Thomas S. BlackerAffiliation 1: Consortium for Mitochondrial Research, Department of Cell & Developmental Biology, University College London, London, United Kingdom
Affiliation 2: Department of Physics & Astronomy, University College London, London, United Kingdom
Bio-protocol author page: a3972
Tunde Berecz
Tunde BereczAffiliation: Consortium for Mitochondrial Research, Department of Cell & Developmental Biology, University College London, London, United Kingdom
Bio-protocol author page: a3973
Michael R. Duchen
Michael R. DuchenAffiliation: Consortium for Mitochondrial Research, Department of Cell & Developmental Biology, University College London, London, United Kingdom
Bio-protocol author page: a3974
 and Gyorgy Szabadkai
Gyorgy SzabadkaiAffiliation 1: Consortium for Mitochondrial Research, Department of Cell & Developmental Biology, University College London, London, United Kingdom
Affiliation 2: Department of Biomedical Sciences, University of Padua and CNR Neuroscience Institute, Padua, Italy
For correspondence: g.szabadkai@ucl.ac.uk
Bio-protocol author page: a3975
date: 1/20/2017, 251 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2105.

[Abstract] NADH and NADPH are redox cofactors, primarily involved in catabolic and anabolic metabolic processes respectively. In addition, NADPH plays an important role in cellular antioxidant defence. In live cells and tissues, the intensity of their spectrally-identical autofluorescence, termed NAD(P)H, can be used to probe the mitochondrial redox state, while ...

In vitro Dephosphorylation Assay of c-Myc

Authors: Peng Liao
Peng LiaoAffiliation: Department of Central Laboratory, Tongji University, Shanghai, China
For correspondence: liaopengchina@gmail.com
Bio-protocol author page: a3718
Weichao Wang
Weichao WangAffiliation: Department of Central Laboratory, Tongji University, Shanghai, China
Bio-protocol author page: a3719
 and Xin Ge
Xin GeAffiliation: Department of Clinical Laboratory Medicine, Shanghai Tenth People’s Hospital of Tongji University, Shanghai, China
Bio-protocol author page: a3720
date: 1/20/2017, 217 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2011.

[Abstract] This protocol describes experimental procedures for in vitro dephosphorylation assay of human protein c-Myc. This protocol can be adapted to detect phosphatase activity of other Ser/Thr phosphatases....

In vivo Efficacy Studies in Cell Line and Patient-derived Xenograft Mouse Models

Authors: Elizabeth A. Tovar
Elizabeth A. TovarAffiliation: Center for Cancer and Cell Biology, Van Andel Research Institute, Grand Rapids, Michigan, USA
Bio-protocol author page: a3966
Curt J. Essenburg
Curt J. EssenburgAffiliation: Center for Cancer and Cell Biology, Van Andel Research Institute, Grand Rapids, Michigan, USA
Bio-protocol author page: a3967
 and Carrie Graveel
Carrie GraveelAffiliation: Center for Cancer and Cell Biology, Van Andel Research Institute, Grand Rapids, Michigan, USA
For correspondence: carrie.graveel@vai.org
Bio-protocol author page: a3968
date: 1/5/2017, 297 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2100.

[Abstract] In vivo xenograft models derived from human cancer cells have been a gold standard for evaluating the genetic drivers of cancer and are valuable preclinical models for evaluating the efficacy of cancer therapeutics. Recently, patient-derived tumorgrafts from multiple tumor types have been developed and shown to more accurately recapitulate the molecular ...
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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, ...

[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 ...

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 ...

[Bio101] Subcutaneous Injection of Tumor Cells

Author: Jason Reuter date: 12/20/2011, 23616 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.166.

[Abstract] Growth of cells in the subcutaneous space of immunocompromised mice is a common method for assaying tumorigenic potential in vivo. This technique is also used to assess the effects of therapeutic interventions on cancer cell lines....

In vitro Tumorsphere Formation Assays

Authors: Sara Johnson
Sara JohnsonAffiliation: Biological Sciences Department, University of South Carolina, Columbia, USA
Bio-protocol author page: a224
Hexin Chen
Hexin ChenAffiliation: Biological Sciences Department, University of South Carolina, Columbia, USA
For correspondence: hchen@biol.sc.edu
Bio-protocol author page: a225
 and Pang-Kuo Lo
Pang-Kuo LoAffiliation: Biological Sciences Department, University of South Carolina, Columbia, USA
Bio-protocol author page: a226
date: 2/5/2013, 23132 views, 3 Q&A
DOI: https://doi.org/10.21769/BioProtoc.325.

[Abstract] A tumorsphere is a solid, spherical formation developed from the proliferation of one cancer stem/progenitor cell. These tumorspheres (Figure 1a) are easily distinguishable from single or aggregated cells (Figure 1b) as the cells appear to become fused together and individual cells cannot be identified. ...

In vivo Matrigel Plug Angiogenesis Assay

Authors: Hong Lok Lung
Hong Lok LungAffiliation: Department of Clinical Oncology and Center for Cancer Research, The University of Hong Kong, Hong Kong , Hong Kong SAR
Bio-protocol author page: a102
 and Maria Li Lung
Maria Li LungAffiliation: Department of Clinical Oncology and Center for Cancer Research, The University of Hong Kong, Hong Kong , Hong Kong SAR
For correspondence: mlilung@hku.hk
Bio-protocol author page: a101
date: 9/20/2012, 22767 views, 2 Q&A
DOI: https://doi.org/10.21769/BioProtoc.261.

[Abstract] The matrigel plug angiogenesis assay is a simple in vivo technique to detect the newly formed blood vessels in the transplanted gel plugs in nude mice. The matrigel matrix is derived from the engelbroth-holm-swarm (EHS) mouse sarcoma, and its composition is comparable to the basement membrane proteins. ...
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