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Georgetown University Medical Center

12 ARTICLES PUBLISHED IN JoVE

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Biology

Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis
Lyuba Khavrutskii 1,2, Joanna Yeh 1, Olga Timofeeva 3, Sergey G. Tarasov 4, Samuel Pritt 1, Karen Stefanisko 1, Nadya Tarasova 1
1Cancer and Inflammation Program, National Cancer Institute, 2Basic Science Program, SAIC-Frederick, Inc., 3Departments of Oncology and Radiation Medicine, Georgetown University Medical Center, 4Structural Biophysics Laboratory, National Cancer Institute

Microscale thermophoresis (MST) can be widely used for determination of binding affinity without purification of the target protein from cell lysates. The protocol involves overexpression of the GFP-fused protein, cell lysis in non-denaturing conditions, and detection of MST signal in the presence of varying concentrations of the ligand.

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Immunology and Infection

Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates
Stefano G. Daniele 1, Amanda A. Edwards 1, Kathleen A. Maguire-Zeiss 1
1Department of Neuroscience, Georgetown University Medical Center

One key to successful investigation of microglial biology is the preservation of microglial immunofunction ex vivo during isolation from CNS tissue. Isolating microglia via rotary shaking results in highly pure and immunofunctional cell cultures as assessed by fluorescent imaging, immunocytochemistry, and ELISA following microglia activation with the proinflammatory stimuli lipopolysaccharide (LPS) and Pam3CSK4 (Pam).

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Bioengineering

Novel Atomic Force Microscopy Based Biopanning for Isolation of Morphology Specific Reagents against TDP-43 Variants in Amyotrophic Lateral Sclerosis
Stephanie M. Williams 1, Lalitha Venkataraman 1, Huilai Tian 1, Galam Khan 2, Brent T. Harris 2,3, Michael R. Sierks 1
1School for Engineering of Matter, Transport and Energy, Arizona State University, 2Department of Neurology, Georgetown University Medical Center, 3Department of Pathology, Georgetown University Medical Center

Using atomic force microscopy in combination with biopanning technology we created a negative and positive biopanning system to acquire antibodies against disease-specific protein variants present in any biological material, even at low concentrations. We were successful in obtaining antibodies to TDP-43 protein variants involved in Amyotrophic Lateral Sclerosis.

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Biology

SpOT the Correct Tissue Every Time in Multi-tissue Blocks
Anna Coffey 1,2, Michael D. Johnson 3, Deborah L. Berry 3
1Center for Advanced Preclinical Research (CAPR), Frederick National Laboratory for Cancer Research, 2Leidos Biomedical Research, Inc., 3Department of Oncology, Georgetown University

The purpose of the Specimen Orientation Tag (SpOT) is to function as an orientation tool to aid in individual tissue identification in multi-tissue paraffin blocks. These protocols demonstrate how it is constructed easily from common, low-cost histology materials and serves as a reliable visual marker in paraffin blocks and sections.

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Medicine

A Cancer Cell Spheroid Assay to Assess Invasion in a 3D Setting
Eric B. Berens 1, Jon M. Holy 2, Anna T. Riegel 1, Anton Wellstein 1
1Lombardi Comprehensive Cancer Center, Department of Oncology, Georgetown University, 2Department of Biomedical Sciences, University of Minnesota

This method evaluates cancer cell invasion from spheroids into a surrounding 3D matrix. Spheroids are generated via the hanging drop culture method and then embedded in a matrix comprised of basement membrane materials and type I collagen. Invasion out of the spheroids is subsequently monitored.

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Cancer Research

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis
Sung-Hyeok Hong *1, Jason U. Tilan *2,3, Susana Galli 1, Rachel Acree 3, Katherine Connors 4, Akanksha Mahajan 1, Larissa Wietlisbach 3, Taylor Polk 3, Ewa Izycka-Swieszewska 5, Yi-Chien Lee 6, Luciane R. Cavalli 6, Olga C. Rodriguez 6, Chris Albanese 6,7, Joanna B. Kitlinska 1
1Department of Biochemistry and Molecular & Cellular Biology, Georgetown University Medical Center, 2Department of Nursing, Georgetown University, School of Nursing and Health Studies, 3Department of Human Science, Georgetown University, School of Nursing and Health Studies, 4School of Medicine, Georgetown University Medical Center, 5Department of Pathology and Neuropathology, Medical University of Gdańsk, 6Department of Oncology, Georgetown University Medical Center, 7Department of Pathology, Georgetown University Medical Center

This manuscript describes the development of an animal model that allows for the direct testing of the effects of tumor hypoxia on metastasis and the deciphering the mechanisms of its action. Although the experiments described here focus on Ewing sarcoma, a similar approach can be applied to other tumor types.

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Cancer Research

Testing the Vascular Invasive Ability of Cancer Cells in Zebrafish (Danio Rerio)
Eric B. Berens 1, Ghada M. Sharif 1, Anton Wellstein 1, Eric Glasgow 1
1Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University

This method utilizes zebrafish embryos to efficiently test the vascular invasive ability of cancer cells. Fluorescent cancer cells are injected into the precardiac sinus or yolk sac of developing embryos. Cancer cell vascular invasion and extravasation is assessed via fluorescence microscopy of the tail region 24 to 96 hr later.

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Biology

Live Cell Imaging and 3D Analysis of Angiotensin Receptor Type 1a Trafficking in Transfected Human Embryonic Kidney Cells Using Confocal Microscopy
Parnika Kadam 1,2, Ryan McAllister 3, Jeffrey S. Urbach 3, Kathryn Sandberg 1,2, Susette C. Mueller 4
1Department of Biochemistry, Georgetown University Medical Center, 2Department of Medicine, Georgetown University Medical Center, 3Department of Physics, Georgetown University Medical Center, 4Department of Oncology, Georgetown University Medical Center

Here we present a protocol to image cells expressing green fluorescent protein-tagged angiotensin type 1a receptors during endocytosis initiated by angiotensin II treatment. This technique includes labeling lysosomes with a second fluorescent marker, and then utilizing software to analyze the co-localization of receptor and lysosome in three dimensions over time.

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Cancer Research

A Rapid Filter Insert-based 3D Culture System for Primary Prostate Cell Differentiation
Lucas Tricoli 1, Deborah L. Berry 2, Chris Albanese 1
1Department of Oncology, Lombardi Comprehensive Cancer Center, 2Department of Oncology, Georgetown University Medical Center

Here, we present a method for the establishment of a rapid in vitro system that supports the three dimensional culturing and subsequent luminal differentiation of primary prostate epithelial cells.

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Medicine

A Mouse Model of Single and Repetitive Mild Traumatic Brain Injury
Bevan S. Main 1, Stephanie S. Sloley 1, Sonia Villapol 1, David N. Zapple 2, Mark P. Burns 1
1Laboratory for Brain Injury and Dementia, Department of Neuroscience, Georgetown University Medical Center, 2University Information Systems, Division of Research Technologies, Georgetown University

Athletes absorb several hundred mild traumatic brain injuries (mTBI)/concussions every year; however, the consequence of these on the brain is poorly understood. Therefore, an animal model of single and repetitive mTBI that consistently replicates clinically relevant symptoms provides the means to advance the study of mTBI and concussion.

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Cancer Research

Monitoring Cancer Cell Invasion and T-Cell Cytotoxicity in 3D Culture
Yuan-Na Lin 1, Apsra Nasir 1, Sharon Camacho 1, Deborah L. Berry 1, Marcel O. Schmidt 1, Gray W. Pearson 1, Anna T. Riegel 1, Anton Wellstein 1
1Lombardi Comprehensive Cancer Center, Department of Oncology, Georgetown University

The presented approach simultaneously evaluates cancer cell invasion in 3D spheroid assays and T-cell cytotoxicity. Spheroids are generated in a scaffold-free agarose multi-microwell cast. Co-culture and embedding in type I collagen matrix are performed within the same device which allows to monitor cancer cell invasion and T-cell mediated cytotoxicity.

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Cancer Research

Real-Time Detection and Capture of Invasive Cell Subpopulations from Co-Cultures
Ghada M. Sharif 1, Leon Der 2, Anna T. Riegel 1, Makarand Paranjape 2, Anton Wellstein 1
1Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, 2Department of Physics, Georgetown University

We describe an approach to detect and capture invasive cell subpopulations in real-time. The experimental design uses Real-Time Cellular Analysis by monitoring changes in the electric impedance of cells. Invasive cancer, immune, endothelial or stromal cells in complex tissues can be captured, and the impact of co-cultures can be assessed.

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