MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression

JoVE 3661 2/17/2012

Mansoureh Sameni¹, Arulselvi Anbalagan¹, Mary B. Olive¹, Kamiar Moin^1,2, Raymond R. Mattingly^1,2, Bonnie F. Sloane^1,2

¹Department of Pharmacology, Wayne State University , ²Barbara Ann Karmanos Cancer Institute, Wayne State University

We have developed 3D coculture models for live-cell imaging in real-time of interactions among breast tumor cells and other cells in their microenvironment that impact progression to an invasive phenotype. These models can serve as preclinical screens for drugs to target paracrine-induced proteolytic, chemokine/cytokine and kinase pathways implicated in invasiveness.

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

JoVE 3860 3/10/2012

Liam O'Ryan, Tracy Rimington, Natasha Cant, Robert C. Ford

Faculty of Life Sciences, University of Manchester

Attempts to express the cystic fibrosis transmembrane conductance regulator (CFTR) in Saccharomyces cerevisiae have, until now, yielded relatively low amounts of protein. This protocol and the associated reagents distributed via the Cystic Fibrosis Foundation should allow the preparation of milligram amounts of this 'difficult' eukaryotic membrane protein.

Screening for Amyloid Aggregation by Semi-Denaturing Detergent-Agarose Gel Electrophoresis

JoVE 838 7/16/2008

Randal Halfmann^1,2,3, Susan Lindquist^1,2,3

¹Whitehead Institute for Biomedical Research, ²Department of Biology, MIT - Massachusetts Institute of Technology, ³Howard Hughes Medical Institute

SDD-AGE is a useful technique for the detection and characterization of amyloid-like polymers in cells. Here we demonstrate an adaptation that makes this technique amenable to large-scale applications.

Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation

JoVE 1792 5/02/2010

Oshri Avraham*, Sophie Zisman*, Yoav Hadas, Lilach Vald, Avihu Klar

Department of Medical Neurobiology, Institute for Medical Research Israel Canada, Hebrew University-Hadassah Medical School

This video demonstrates how to visualize axonal pathways of genetically defined groups of neurons in the embryonic chick spinal cord utilizing in ovo electroporation of reporter genes under the control of specific enhancer elements.

The 2009 Lindau Nobel Laureate Meeting: Aaron Ciechanover, Chemistry 2004

JoVE 1559 7/01/2009

Aaron Ciechanover

Aaron Ciechanover was born in Haifa, Israel in October 1947. Ciechanover shared the Nobel Prize in Chemistry in 2004 with Avram Hershko and Irwin Rose for their discovery of ubiquitin-mediated protein degradation.

Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

JoVE 3370 1/16/2012

Johan Nilvebrant, Tove Alm, Sophia Hober

School of Biotechnology, Department of Proteomics, Royal Institute of Technology

A novel and highly efficient two-step affinity chromatography protocol has been developed and is described in detail. The method is based on a small purification tag with two inherent affinities and is applicable to a wide range of target proteins with different properties.

Modeling and Imaging 3-Dimensional Collective Cell Invasion

JoVE 3525 12/07/2011

Rebecca W. Scott¹, Diane Crighton², Michael F. Olson²

¹Strathclyde Institute for Pharmacy and Biomedical Sciences, University of Strathclyde , ²The Beatson Institute for Cancer Research

Models of tumor cell invasion into three-dimensional extracellular matrix better reflect the in vivo situation than two-dimensional motility assays. Using matrix invasion assays combined with confocal imaging of fluorescently-labeled cells, detailed information on invasion modes and the distinct contributions of leading versus following cells can be obtained.

Western Blotting: Sample Preparation to Detection

JoVE 2359 10/14/2010

Anna Eslami, Jesse Lujan

Research and Development, EMD Chemicals Inc.

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract.

Immuno-fluorescence Assay of Leptospiral Surface-exposed Proteins

JoVE 2805 7/01/2011

Marija Pinne^1,2, David Haake^3,4

¹Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, ²Research service, 151, Veterans Affairs Greater Los Angeles Healthcare System, ³Departments of Medicine, Urology at David Geffen School of Medicine and Department of Microbiology, Immunology and Molecular Gentics, University of California Los Angeles (UCLA), ⁴Division of Infectious Diseases, 111F, Veterans Affairs Greater Los Angeles Health Care System

An efficient method to assess surface-exposure of leptospiral proteins is described. The method is specifically designed to avoid disruption of the fragile outer membrane of leptospiral cells. This technique requires employment of several negative controls to assess the integrity of the outer membrane and specificity of antibody reaction.

A Method for Ovarian Follicle Encapsulation and Culture in a Proteolytically Degradable 3 Dimensional System

JoVE 2695 3/15/2011

Ariella Shikanov¹, Min Xu², Teresa K. Woodruff^2,3,4, Lonnie D. Shea^1,4,5

¹Institute for BioNanotechnology in Advanced Medicine, Northwestern University, ²Department of Obstetrics and Gynecology, Northwestern University, Feinberg School of Medicine, ³Center for Reproductive Research, Northwestern University, ⁴The Robert H. Lurie Comprehensive Cancer Center, Northwestern University, ⁵Department of Chemical and Biological Engineering, Northwestern University

A new method for ovarian follicle encapsulation in a 3D fibrin-alginate interpenetrating network is described. This system combines structural support with proteolytic degradation to support the development of immature follicles to produce mature oocytes. This method may be applied to culture cell aggregates to maintain cell-cell contacts without limiting expansion.