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Cancer Biology

Isolation of Murine Alveolar Type II Epithelial Cells

Featured protocol,  Authors: Fan Sun
Fan SunAffiliation 1: University of Pittsburgh Cancer Institute, University of Pittsburgh, Pittsburgh, USA
Affiliation 2: Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, USA
Bio-protocol author page: a4527
Gutian Xiao
Gutian XiaoAffiliation 1: University of Pittsburgh Cancer Institute, University of Pittsburgh, Pittsburgh, USA
Affiliation 2: Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, USA
Bio-protocol author page: a4528
 and Zhaoxia Qu
Zhaoxia QuAffiliation 1: University of Pittsburgh Cancer Institute, University of Pittsburgh, Pittsburgh, USA
Affiliation 2: Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, USA
For correspondence: quz@upmc.edu
Bio-protocol author page: a4529
date: 5/20/2017, 125 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2288.

Brief version appeared in Oncogene, May 2016
We have optimized a protocol for isolation of alveolar type II epithelial cells from mouse lung. Lung cell suspensions are prepared by intratracheal instillation of dispase and agarose followed by mechanical disaggregation of the lungs. Alveolar type II epithelial cells are purified from these lung cell suspensions through magnetic-based negative selection using a Biotin-antibody, Streptavidin-MicroBeads system. The purified alveolar type II epithelial cells can be cultured and maintained on fibronectin-coated plates in DMEM with 10% FBS. This protocol enables specific investigation of alveolar type II epithelial cells at molecular and cellular levels and provides an important tool to investigate in vitro the mechanisms underlying lung pathogenesis.

Murine Bronchoalveolar Lavage

Featured protocol,  Authors: Fan Sun
Fan SunAffiliation 1: University of Pittsburgh Cancer Institute, University of Pittsburgh, Pittsburgh, USA
Affiliation 2: Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, USA
Bio-protocol author page: a4527
Gutian Xiao
Gutian XiaoAffiliation 1: University of Pittsburgh Cancer Institute, University of Pittsburgh, Pittsburgh, USA
Affiliation 2: Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, USA
Bio-protocol author page: a4528
 and Zhaoxia Qu
Zhaoxia QuAffiliation 1: University of Pittsburgh Cancer Institute, University of Pittsburgh, Pittsburgh, USA
Affiliation 2: Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, USA
For correspondence: quz@upmc.edu
Bio-protocol author page: a4529
date: 5/20/2017, 117 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2287.

Brief version appeared in Oncogene, May 2016
A basic Bronchoalveolar lavage (BAL) procedure in mouse is described here. Cells and fluids obtained from BAL can be analyzed by Hema3-staining, immunostaining, Fluorescence-activated cell sorting (FACS), PCR, bicinchoninic acid protein assay, enzyme-linked immunosorbent assay (ELISA), luminex assays, etc., to examine the immune cells, pathogens, proteins such as cytokines/chemokines, and the expression levels of inflammation-related and other genes in the cells. This will help to understand the underlying mechanisms of these lung diseases and develop specific and effective drugs.

Analysis of in vivo Interaction between RNA Binding Proteins and Their RNA Targets by UV Cross-linking and Immunoprecipitation (CLIP) Method

Featured protocol,  Authors: Pamela Bielli
Pamela BielliAffiliation 1: Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy
Affiliation 2: Laboratory of Neuroembriology, Fondazione Santa Lucia, Rome, Italy
Bio-protocol author page: a4492
 and Claudio Sette
Claudio SetteAffiliation 1: Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy
Affiliation 2: Laboratory of Neuroembriology, Fondazione Santa Lucia, Rome, Italy
For correspondence: claudio.sette@uniroma2.it
Bio-protocol author page: a4493
date: 5/20/2017, 152 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2274.

Brief version appeared in Oncogene, Apr 2016
RNA metabolism is tightly controlled across different tissues and developmental stages, and its dysregulation is one of the molecular hallmarks of cancer. Through direct binding to specific sequence element(s), RNA binding proteins (RBPs) play a pivotal role in co- and post-transcriptional RNA regulatory events. We have recently demonstrated that, in pancreatic cancer cells, acquisition of a drug resistant (DR)-phenotype relied on upregulation of the polypyrimidine tract binding protein (PTBP1), which in turn is recruited to the pyruvate kinase pre-mRNA and favors splicing of the oncogenic PKM2 variant. Herein, we describe a step-by-step protocol of the ultraviolet (UV) light cross-linking and immunoprecipitation (CLIP) method to determine the direct binding of a RBP to specific regions of its target RNAs in adherent human cell lines.

Nucleosome Positioning Assay

Featured protocol,  Authors: Zhongliang Zhao
Zhongliang ZhaoAffiliation: Division of Molecular Biology of the Cell II, German Cancer Research Center, DKFZ-ZMBH Alliance, Im Neuenheimer Feld 581, 69120 Heidelberg, Germany
Bio-protocol author page: a4525
 and Holger Bierhoff
Holger BierhoffAffiliation 1: Department of Biochemistry, Institute of Biochemistry and Biophysics, Center for Molecular Biomedicine (CMB), Friedrich Schiller University Jena, Hans-Knöll-Str. 2, Jena, Germany
Affiliation 2: Leibniz-Institute on Aging – Fritz Lipmann Institute (FLI), Beutenbergstrasse 11, Jena, Germany
For correspondence: holger.bierhoff@uni-jena.de
Bio-protocol author page: a4526
date: 5/20/2017, 102 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2285.

Brief version appeared in Cell Rep, Mar 2016
The basic unit of chromatin is the nucleosome, a histone octamer with 147 base pairs of DNA wrapped around it. Positions of nucleosomes relative to each other and to DNA elements have a strong impact on chromatin structure and gene activity and are tightly regulated at multiple levels, i.e., DNA sequence, transcription factor binding, histone modifications and variants, and chromatin remodeling enzymes (Bell et al., 2011; Hughes and Rando, 2014). Nucleosome positions in cells or isolated nuclei can be detected by partial nuclease digestion of native or cross-linked chromatin followed by ligation-mediated polymerase chain reaction (LM-PCR) (McPherson et al., 1993; Soutoglou and Talianidis, 2002). This protocol describes a nucleosome positioning assay using Micrococcal Nuclease (MNase) digestion of formaldehyde-fixed chromatin followed by LM-PCR. We exemplify the nucleosome positioning assay for the promoter of genes encoding ribosomal RNA (rRNA genes or rDNA) in mice, which has two mutually exclusive configurations. The rDNA promoter harbors either an upstream nucleosome (NucU) covering nucleotides -157 to -2 relative to the transcription start site, or a downstream nucleosome (NucD) at position -132 to +22 (Li et al., 2006; Xie et al., 2012). Radioactive labeling of LM-PCR products followed by denaturing urea-polyacrylamide gel electrophoresis allows resolution and relative quantification of both configurations. As depicted in the diagram in Figure 1, the nucleosome positioning assay is a versatile low to medium throughput method to map discrete nucleosome positions with high precision in a semi-quantitative manner.

Imaging the Pharynx to Measure the Uptake of Doxorubicin in Caenorhabditis elegans

Featured protocol,  Authors: Sivathevy Amirthagunabalasingam
Sivathevy AmirthagunabalasingamAffiliation: Maisonneuve-Rosemont Hospital Research Center, and the Université de Montréal, Faculty of Medicine, Department of Medicine, Montréal, Canada
Bio-protocol author page: a4534
Arturo Papaluca
Arturo PapalucaAffiliation: , Lady Davis Institute, McGill University, Montréal, Canada
Bio-protocol author page: a4535
Taramatti Harihar
Taramatti HariharAffiliation: Maisonneuve-Rosemont Hospital Research Center, and the Université de Montréal, Faculty of Medicine, Department of Medicine, Montréal, Canada
Bio-protocol author page: a4536
 and Dindial Ramotar
Dindial RamotarAffiliation: Maisonneuve-Rosemont Hospital Research Center, and the Université de Montréal, Faculty of Medicine, Department of Medicine, Montréal, Canada
For correspondence: dindial.ramotar@umontreal.ca
Bio-protocol author page: a4537
date: 5/20/2017, 98 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2291.

Brief version appeared in Sci Rep, Oct 2016
Caenorhabditis elegans offers an array of advantages to investigate the roles of uptake transporters. Herein, an epifluorescent microscopy approach was developed to monitor the uptake of the autofluorescent anticancer drug, doxorubicin, into the pharynx of C. elegans by organic cation transporters.

Semi-quantitative Analysis of H4K20me1 Levels in Living Cells Using Mintbody

Featured protocol,  Authors: Yuko Sato
Yuko SatoAffiliation: Cell Biology Unit, Institute of Innovative Research, Tokyo Institute of Technology, Yokohama, Japan
For correspondence: satoy@bio.titech.ac.jp
Bio-protocol author page: a4494
 and Hiroshi Kimura
Hiroshi KimuraAffiliation: Cell Biology Unit, Institute of Innovative Research, Tokyo Institute of Technology, Yokohama, Japan
For correspondence: hkimura@bio.titech.ac.jp
Bio-protocol author page: a4495
date: 5/20/2017, 138 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2276.

Brief version appeared in J Mol Biol, Oct 2016
Eukaryotic nuclear DNA wraps around histone proteins to form a nucleosome, a basic unit of chromatin. Posttranslational modification of histones play an important role in gene regulation and chromosome duplication. Some modifications are quite stable to be an epigenetic memory, and others exhibit rapid turnover or fluctuate during the cell cycle. Histone H4 Lys20 monomethylation (H4K20me1) has been shown to be involved in chromosome condensation, segregation, replication and repair. H4K20 methylation is controlled through a few methyltransferases, PR-Set7/Set8, SUV420H1, and SUV420H2, and a demethylase, PHF8. In cycling cells, the level of H4K20me1 increases during G2 and M phases and decreases during G1 phase. To monitor the local concentration and global fluctuation of histone modifications in living cells, we have developed a genetically encoded probe termed mintbody (modification-specific intracellular antibody; Sato et al., 2013 and 2016). By measuring the nuclear to cytoplasmic intensity ratio, the relative level of H4K20me1 in individual cells can be monitored. This detailed protocol allows the semi-quantitative analysis of the effects of methyltransferases on H4K20me1 levels in living cells based on H4K20me1-mintbody described by Sato et al. (2016).

Immunostaining of Formaldehyde-fixed Metaphase Chromosome from Untreated and Aphidicolin-treated DT40 Cells

Featured protocol,  Author: Vibe H. Oestergaard
Vibe H. OestergaardAffiliation: Department of Biology, University of Copenhagen, Ole Maaloees Vej 5, Copenhagen N, Denmark
For correspondence: vibe@bio.ku.dk
Bio-protocol author page: a4458
date: 5/5/2017, 175 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2259.

Brief version appeared in J Cell Biol, Aug 2015
During mitosis chromosomes are condensed into dense X-shaped structures that allow for microscopic determination of karyotype as well as inspection of chromosome morphology.

Relative Stiffness Measurements of Tumour Tissues by Shear Rheology

Featured protocol,  Authors: Chris D. Madsen
Chris D. MadsenAffiliation: Department of Laboratory Medicine, Division of Translational Cancer Research, Lund University, Lund, Sweden
For correspondence: chris.madsen@med.lu.se
Bio-protocol author page: a3987
 and Thomas R. Cox
Thomas R. CoxAffiliation: The Garvan Institute of Medical Research and The Kinghorn Cancer Centre, Cancer Division, St Vincent's Clinical School, Faculty of Medicine, UNSW Sydney, Australia
For correspondence: t.cox@garvan.org.au
Bio-protocol author page: a3986
date: 5/5/2017, 236 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2265.

Brief version appeared in EMBO Rep, Oct 2015
The microenvironment of solid tumours is a critical contributor to the progression of tumours and offers a promising target for therapeutic intervention (Cox and Erler, 2011; Barker et al., 2012; Cox et al., 2016; Cox and Erler, 2016). The properties of the tumour microenvironment vary significantly from that of the original tissue in both biochemistry and biomechanics. At present, the complex interplay between the biomechanical properties of the microenvironment and tumour cell phenotype are under intense investigation. The ability to measure the biomechanical properties of tumour samples from cancer models will increase our understanding of their importance in solid tumour biology. Here we report a simple method to measure the viscoelastic properties of tumour specimens using a controlled strain rotational rheometer.

Isolation of Murine Alveolar Type II Epithelial Cells

Authors: Fan Sun
Fan SunAffiliation 1: University of Pittsburgh Cancer Institute, University of Pittsburgh, Pittsburgh, USA
Affiliation 2: Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, USA
Bio-protocol author page: a4527
Gutian Xiao
Gutian XiaoAffiliation 1: University of Pittsburgh Cancer Institute, University of Pittsburgh, Pittsburgh, USA
Affiliation 2: Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, USA
Bio-protocol author page: a4528
 and Zhaoxia Qu
Zhaoxia QuAffiliation 1: University of Pittsburgh Cancer Institute, University of Pittsburgh, Pittsburgh, USA
Affiliation 2: Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, USA
For correspondence: quz@upmc.edu
Bio-protocol author page: a4529
date: 5/20/2017, 125 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2288.

[Abstract] We have optimized a protocol for isolation of alveolar type II epithelial cells from mouse lung. Lung cell suspensions are prepared by intratracheal instillation of dispase and agarose followed by mechanical disaggregation of the lungs. Alveolar type II epithelial cells are purified from these lung cell suspensions through magnetic-based negative selection ...

Murine Bronchoalveolar Lavage

Authors: Fan Sun
Fan SunAffiliation 1: University of Pittsburgh Cancer Institute, University of Pittsburgh, Pittsburgh, USA
Affiliation 2: Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, USA
Bio-protocol author page: a4527
Gutian Xiao
Gutian XiaoAffiliation 1: University of Pittsburgh Cancer Institute, University of Pittsburgh, Pittsburgh, USA
Affiliation 2: Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, USA
Bio-protocol author page: a4528
 and Zhaoxia Qu
Zhaoxia QuAffiliation 1: University of Pittsburgh Cancer Institute, University of Pittsburgh, Pittsburgh, USA
Affiliation 2: Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, USA
For correspondence: quz@upmc.edu
Bio-protocol author page: a4529
date: 5/20/2017, 117 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2287.

[Abstract] A basic Bronchoalveolar lavage (BAL) procedure in mouse is described here. Cells and fluids obtained from BAL can be analyzed by Hema3-staining, immunostaining, Fluorescence-activated cell sorting (FACS), PCR, bicinchoninic acid protein assay, enzyme-linked immunosorbent assay (ELISA), luminex assays, etc., to examine the immune cells, pathogens, proteins ...

Analysis of in vivo Interaction between RNA Binding Proteins and Their RNA Targets by UV Cross-linking and Immunoprecipitation (CLIP) Method

Authors: Pamela Bielli
Pamela BielliAffiliation 1: Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy
Affiliation 2: Laboratory of Neuroembriology, Fondazione Santa Lucia, Rome, Italy
Bio-protocol author page: a4492
 and Claudio Sette
Claudio SetteAffiliation 1: Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy
Affiliation 2: Laboratory of Neuroembriology, Fondazione Santa Lucia, Rome, Italy
For correspondence: claudio.sette@uniroma2.it
Bio-protocol author page: a4493
date: 5/20/2017, 152 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2274.

[Abstract] RNA metabolism is tightly controlled across different tissues and developmental stages, and its dysregulation is one of the molecular hallmarks of cancer. Through direct binding to specific sequence element(s), RNA binding proteins (RBPs) play a pivotal role in co- and post-transcriptional RNA regulatory events. We have recently demonstrated that, ...

Nucleosome Positioning Assay

Authors: Zhongliang Zhao
Zhongliang ZhaoAffiliation: Division of Molecular Biology of the Cell II, German Cancer Research Center, DKFZ-ZMBH Alliance, Im Neuenheimer Feld 581, 69120 Heidelberg, Germany
Bio-protocol author page: a4525
 and Holger Bierhoff
Holger BierhoffAffiliation 1: Department of Biochemistry, Institute of Biochemistry and Biophysics, Center for Molecular Biomedicine (CMB), Friedrich Schiller University Jena, Hans-Knöll-Str. 2, Jena, Germany
Affiliation 2: Leibniz-Institute on Aging – Fritz Lipmann Institute (FLI), Beutenbergstrasse 11, Jena, Germany
For correspondence: holger.bierhoff@uni-jena.de
Bio-protocol author page: a4526
date: 5/20/2017, 102 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2285.

[Abstract] The basic unit of chromatin is the nucleosome, a histone octamer with 147 base pairs of DNA wrapped around it. Positions of nucleosomes relative to each other and to DNA elements have a strong impact on chromatin structure and gene activity and are tightly regulated at multiple levels, i.e., DNA sequence, transcription factor binding, histone modifications ...

Imaging the Pharynx to Measure the Uptake of Doxorubicin in Caenorhabditis elegans

Authors: Sivathevy Amirthagunabalasingam
Sivathevy AmirthagunabalasingamAffiliation: Maisonneuve-Rosemont Hospital Research Center, and the Université de Montréal, Faculty of Medicine, Department of Medicine, Montréal, Canada
Bio-protocol author page: a4534
Arturo Papaluca
Arturo PapalucaAffiliation: , Lady Davis Institute, McGill University, Montréal, Canada
Bio-protocol author page: a4535
Taramatti Harihar
Taramatti HariharAffiliation: Maisonneuve-Rosemont Hospital Research Center, and the Université de Montréal, Faculty of Medicine, Department of Medicine, Montréal, Canada
Bio-protocol author page: a4536
 and Dindial Ramotar
Dindial RamotarAffiliation: Maisonneuve-Rosemont Hospital Research Center, and the Université de Montréal, Faculty of Medicine, Department of Medicine, Montréal, Canada
For correspondence: dindial.ramotar@umontreal.ca
Bio-protocol author page: a4537
date: 5/20/2017, 98 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2291.

[Abstract] Caenorhabditis elegans offers an array of advantages to investigate the roles of uptake transporters. Herein, an epifluorescent microscopy approach was developed to monitor the uptake of the autofluorescent anticancer drug, doxorubicin, into the pharynx of C. elegans by organic cation transporters....

Semi-quantitative Analysis of H4K20me1 Levels in Living Cells Using Mintbody

Authors: Yuko Sato
Yuko SatoAffiliation: Cell Biology Unit, Institute of Innovative Research, Tokyo Institute of Technology, Yokohama, Japan
For correspondence: satoy@bio.titech.ac.jp
Bio-protocol author page: a4494
 and Hiroshi Kimura
Hiroshi KimuraAffiliation: Cell Biology Unit, Institute of Innovative Research, Tokyo Institute of Technology, Yokohama, Japan
For correspondence: hkimura@bio.titech.ac.jp
Bio-protocol author page: a4495
date: 5/20/2017, 138 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2276.

[Abstract] Eukaryotic nuclear DNA wraps around histone proteins to form a nucleosome, a basic unit of chromatin. Posttranslational modification of histones play an important role in gene regulation and chromosome duplication. Some modifications are quite stable to be an epigenetic memory, and others exhibit rapid turnover or fluctuate during the cell cycle. Histone ...

Immunostaining of Formaldehyde-fixed Metaphase Chromosome from Untreated and Aphidicolin-treated DT40 Cells

Author: Vibe H. Oestergaard
Vibe H. OestergaardAffiliation: Department of Biology, University of Copenhagen, Ole Maaloees Vej 5, Copenhagen N, Denmark
For correspondence: vibe@bio.ku.dk
Bio-protocol author page: a4458
date: 5/5/2017, 175 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2259.

[Abstract] During mitosis chromosomes are condensed into dense X-shaped structures that allow for microscopic determination of karyotype as well as inspection of chromosome morphology.
   This protocol describes a method to perform immunostaining of formaldehyde-fixed metaphase chromosomes from the avian cell line DT40. It was developed to characterize the localization ...

Relative Stiffness Measurements of Tumour Tissues by Shear Rheology

Authors: Chris D. Madsen
Chris D. MadsenAffiliation: Department of Laboratory Medicine, Division of Translational Cancer Research, Lund University, Lund, Sweden
For correspondence: chris.madsen@med.lu.se
Bio-protocol author page: a3987
 and Thomas R. Cox
Thomas R. CoxAffiliation: The Garvan Institute of Medical Research and The Kinghorn Cancer Centre, Cancer Division, St Vincent's Clinical School, Faculty of Medicine, UNSW Sydney, Australia
For correspondence: t.cox@garvan.org.au
Bio-protocol author page: a3986
date: 5/5/2017, 236 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2265.

[Abstract] The microenvironment of solid tumours is a critical contributor to the progression of tumours and offers a promising target for therapeutic intervention (Cox and Erler, 2011; Barker et al., 2012; Cox et al., 2016; Cox and Erler, 2016). The properties of the tumour microenvironment vary significantly from that of the original tissue in both biochemistry ...

Virtual Screening of Transmembrane Serine Protease Inhibitors

Authors: Antti Poso*
Antti PosoAffiliation 1: School of Pharmacy, University of Eastern Finland, Kuopio, Finland
Affiliation 2: Department of Internal Medicine VIII, University Hospital Tübingen, Tübingen, Germany
Bio-protocol author page: a4406
Topi Tervonen*
Topi TervonenAffiliation: Cancer Cell Circuitry Laboratory, Research Programs Unit/Translational Cancer Biology, University of Helsinki, Helsinki, Finland
Bio-protocol author page: a4407
 and Juha Klefström*
Juha KlefströmAffiliation: Cancer Cell Circuitry Laboratory, Research Programs Unit/Translational Cancer Biology, University of Helsinki, Helsinki, Finland
For correspondence: juha.klefstrom@helsinki.fi
Bio-protocol author page: a3835
 (*contributed equally to this work) date: 4/20/2017, 345 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2246.

[Abstract] The human family of type II transmembrane serine proteases includes 17 members. The defining features of these proteases are an N-terminal transmembrane domain and a C-terminal serine protease of the chymotrypsin (S1) fold, separated from each other by a variable stem region. Recently accumulated evidence suggests a critical role for these proteases ...

Melanoma Stem Cell Sphere Formation Assay

Authors: Alessandra Tuccitto
Alessandra TuccittoAffiliation: Department of Experimental Oncology and Molecular Medicine, Unit of Immunotherapy of Human Tumors, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy
Bio-protocol author page: a4374
Valeria Beretta
Valeria BerettaAffiliation: Department of Experimental Oncology and Molecular Medicine, Unit of Immunotherapy of Human Tumors, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy
Bio-protocol author page: a4375
Francesca Rini
Francesca RiniAffiliation: Department of Experimental Oncology and Molecular Medicine, Unit of Immunotherapy of Human Tumors, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy
Bio-protocol author page: a4376
Chiara Castelli
Chiara CastelliAffiliation: Department of Experimental Oncology and Molecular Medicine, Unit of Immunotherapy of Human Tumors, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy
For correspondence: chiara.castelli@istitutotumori.mi.it
Bio-protocol author page: a4377
 and Michela Perego
Michela PeregoAffiliation: The Wistar Institute, Philadelphia, USA
For correspondence: mperego@wistar.org
Bio-protocol author page: a4378
date: 4/20/2017, 292 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2233.

[Abstract] Self-renewal is the ability of cells to replicate themselves at every cell cycle. Throughout self-renewal in normal tissue homeostasis, stem cell number is maintained constant throughout life. Cancer stem cells (CSCs) share this ability with normal tissue stem cells and the sphere formation assay (SFA) is the gold standard assay to assess stem cells ...
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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, 55889 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, 43688 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, 39658 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, 38893 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, 33019 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, 31670 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, 29123 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, 25573 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, 24985 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, 23951 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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