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University of Nebraska, Lincoln

8 ARTICLES PUBLISHED IN JoVE

Biology

A Technique to Simultaneously Visualize Virus-Specific CD8+ T Cells and Virus-Infected Cells In situ

Qingsheng Li¹, Pamela J. Skinner², Lijie Duan¹, Ashley T. Haase¹

¹Department of Microbiology, Medical School, University of Minnesota, ²Department of Veterinary and Biomedical Sciences, University of Minnesota

A technique combining in situ tetramer staining and in situ hybridization (ISTH) enables visualization, mapping and analysis of the spatial proximity of virus-specific CD8+ T cells to their virus-infected targets, and determination of the quantitative relationships between these immune effectors and targets to infection outcomes.

Biology

In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

Sandhya Gopalakrishnan¹, Edward N. Harris¹

¹Department of Biochemistry, University of Nebraska, Lincoln

The study of liver sinusoidal endothelial cells (SECs) must be performed with primary cells obtained from the animal as no cell lines exist. This method relies on liver digestion and differential centrifugation for SEC purification for subsequent culturing and experimentation.

Medicine

Noninvasive Assessment of Cardiac Abnormalities in Experimental Autoimmune Myocarditis by Magnetic Resonance Microscopy Imaging in the Mouse

Chandirasegaran Massilamany^*1, Vahid Khalilzad-Sharghi^*2, Arunakumar Gangaplara¹, David Steffen¹, Shadi F. Othman², Jay Reddy¹

¹School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln, ²Department of Biological Systems Engineering, University of Nebraska-Lincoln

This study demonstrates the successful establishment of magnetic resonance microscopy imaging as a non-invasive tool to assess the cardiac abnormalities in mice affected with autoimmune myocarditis. The data indicate that the technique can be used to monitor the disease-progression in live animals.

Immunology and Infection

In Situ Detection of Autoreactive CD4 T Cells in Brain and Heart Using Major Histocompatibility Complex Class II Dextramers

Chandirasegaran Massilamany¹, Arunakumar Gangaplara¹, Ting Jia¹, Christian Elowsky², Qingsheng Li³, You Zhou², Jay Reddy¹

¹School of Veterinary Medicine and Biomedical Sciences, University of Nebraska, Lincoln, ²Center for Biotechnology, University of Nebraska, Lincoln, ³Nebraska Center for Virology and School of Biological Sciences, University of Nebraska, Lincoln

The protocol to detect self-reactive CD4 T cells in brain and heart by direct staining with major histocompatibility complex class II dextramers has been described in this report. For comprehensive analysis, a reliable method to enumerate the frequencies of antigen-specific CD4⁺ T cells in situ is also devised.

Biochemistry

Purification of Hepatocytes and Sinusoidal Endothelial Cells from Mouse Liver Perfusion

Fatima Cabral¹, Colton M. Miller¹, Katrina M. Kudrna¹, Blake E. Hass¹, Jocelyn G. Daubendiek¹, Brianna M. Kellar¹, Edward N. Harris¹

¹Department of Biochemistry, University of Nebraska

The goal of this protocol is to obtain high viability and high yield of hepatocytes and sinusoidal endothelial cells from liver. This is accomplished by perfusing the liver with a type IV collagenase solution via the portal vein, followed by differential centrifugation to obtain hepatocytes and sinusoidal endothelial cells.

Biochemistry

A Double Humanized BLT-mice Model Featuring a Stable Human-Like Gut Microbiome and Human Immune System

Lance Daharsh^1,2, Jianshui Zhang^1,2, Amanda Ramer-Tait³, Qingsheng Li^1,2

¹Nebraska Center for Virology, ²School of Biological Sciences, University of Nebraska-Lincoln, ³Department of Food Science and Technology, University of Nebraska-Lincoln

We describe a novel method for generating double humanized BLT-mice that feature a functional human immune system and a stable engrafted human-like gut microbiome. This protocol can be followed without the need for germ-free mice or gnotobiotic facilities.

Biochemistry

Determining Total Protein and Bioactive Protein Concentrations in Bovine Colostrum

Ayşenur Arslan¹, Hatice Duman¹, Merve Kaplan¹, Hasan Uzkuç¹, Ayşe Bayraktar^1,2, Melih Ertürk², Merve Alkan^1,2, Steven A. Frese^3,4, Rebbeca M. Duar⁵, Bethany M. Henrick^4,5, Sercan Karav¹

¹Department of Molecular Biology and Genetics, Çanakkale Onsekiz Mart University, ²Uluova Dairy Farm, ³Department of Nutrition, University of Nevada, Reno, ⁴Department of Food Science and Technology, University of Nebraska, Lincoln, ⁵Evolve BioSystems Inc.

Bovine colostrum is both a primary source of nutrients and immunological support for the newborn calf. The understanding of the level of therapeutic proteins (lactoferrin and IgG) is important for the bovine colostrum dosing and standardization for human consumption.

Bioengineering

Direct-Contact Co-culture of Astrocytes and Glioblastoma Cells Patterned using Polyelectrolyte Multilayer Templates

Kimberly M. Stanke^1,2, Srivatsan Kidambi^{1,2,4,5,6,7,8}

¹Complex Biosystems Graduate Program, University of Nebraska, Lincoln, ²Department of Chemical and Biomolecular Engineering, University of Nebraska, Lincoln, ³Nebraska Redox Biology Center, University of Nebraska, Lincoln, ⁴Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center, ⁵Nebraska Center for Integrated Biomolecular Communication, University of Nebraska, Lincoln, ⁶Nebraska Center for the Prevention of Obesity Diseases, University of Nebraska, Lincoln, ⁷Nebraska Center for Materials and Nanoscience, University of Nebraska, Lincoln, ⁸Mary and Dick Holland Regenerative Medicine Program, University of Nebraska Medical Center

This protocol describes a patterned direct contact glioma-astrocyte co-culture utilizing micro-contact printing on polyelectrolyte multilayers (PEMs) to pattern U87 or A172 GBM cells and primary astrocytes.