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Brigham and Women's Hospital and Harvard Medical School

10 ARTICLES PUBLISHED IN JoVE

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Biology

Isolation of Early Hematopoietic Stem Cells from Murine Yolk Sac and AGM
Kelly Morgan 1, Michael Kharas 1, Elaine Dzierzak 2, D. Gary Gilliland 1,3
1Department of Hematology and Oncology, Brigham and Women's Hospital and Harvard Medical School, 2Department of Cell Biology and Genetics, Erasmus University Medical Center, 3Department of Medicine, Howard Hughes Medical Institute, Brigham and Women's Hospital and Harvard Medical School

This video shows how to micro-dissect the yolk sac and aorta-gonad-mesonephros region from embryos and use flow cytometry to sort hematopoietic stem cells.

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Biology

Analysis of Physiologic E-Selectin-Mediated Leukocyte Rolling on Microvascular Endothelium
Georg Wiese 1, Steven R. Barthel 2, Charles J. Dimitroff 2
1Department of Dermatology, Brigham and Women's Hospital, 2Department of Dermatology, Brigham and Women's Hospital and Harvard Medical School

This report provides a visual depiction of parallel-plate flow chamber analysis for studying leukocyte endothelial interactions under physiologic shear stress. This method is particularly useful for investigating the role of endothelial (E)-selectin and leukocyte E-selectin ligands that trigger leukocyte rolling on endothelial cell surfaces.

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Bioengineering

Studying Cell Rolling Trajectories on Asymmetric Receptor Patterns
Chia-Hua Lee 1, Suman Bose 2, Krystyn J. Van Vliet 1, Jeffrey M. Karp 3, Rohit Karnik 2
1Department of Materials Science and Engineering, MIT - Massachusetts Institute of Technology, 2Department of Mechanical Engineering, MIT - Massachusetts Institute of Technology, 3HST Center for Biomedical Engineering and Harvard Stem Cell Institute, Brigham and Women's Hospital and Harvard Medical School

We describe a protocol to observe and analyze cell rolling trajectories on asymmetric receptor-patterned substrates. The resulting data are useful for engineering of receptor-patterned substrates for label-free cell separation and analysis.

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Immunology and Infection

Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation
Tatiana Veremeyko 1, Sarah-Christine Starossom 1, Howard L. Weiner 1, Eugene D. Ponomarev 1
1Center for Neurologic Diseases, Brigham and Women's Hospital, Harvard Medical School

Microglia are resident macrophages that provide the first line of defense and immune surveillance of the central nervous system. MicroRNAs are regulatory molecules that play an important role in many physiological processes including activation and differentiation of macrophages. In this article, we describe the method for measurement of microRNAs in microglia.

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Bioengineering

Systematic Analysis of In Vitro Cell Rolling Using a Multi-well Plate Microfluidic System
Oren Levy 1,2,3,4,5, Priya Anandakumaran 1,2,3,4,5, Jessica Ngai 1,2,3,4,5, Rohit Karnik 6, Jeffrey M. Karp 1,2,3,4,5
1Division of Biomedical Engineering, Department of Medicine, Brigham and Women's Hospital, 2Center for Regenerative Therapeutics, Brigham and Women's Hospital, 3Harvard Medical School, Harvard University, 4Harvard Stem Cell Institute, Harvard University, 5Harvard-MIT Division of Health Sciences and Technology, 6Department of Mechanical Engineering, Massachusetts Institute of Technology

This study used a multi-well plate microfluidic system, significantly increasing throughput of cell rolling studies under physiologically relevant shear flow. Given the importance of cell rolling in the multi-step cell homing cascade and the importance of cell homing following systemic delivery of exogenous populations of cells in patients, this system offers potential as a screening platform to improve cell-based therapy.

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Bioengineering

Isolation and Characterization Of Chimeric Human Fc-expressing Proteins Using Protein A Membrane Adsorbers And A Streamlined Workflow
Monica M. Burdick 1,2, Nathan M. Reynolds 1,2, Eric W. Martin 2, Jacquelyn V. Hawes 1, Grady E. Carlson 1, Chaz M. Cuckler 1, Michael C. Bates 1, Steven R. Barthel 3, Charles J. Dimitroff 3
1Department of Chemical and Biomolecular Engineering, Ohio University, 2Biomedical Engineering Program, Russ College of Engineering and Technology, Ohio University, 3Department of Dermatology, Brigham and Women's Hospital, Harvard Medical School

Compared with traditional affinity chromatography using protein A agarose bead-packed columns, protein A membrane adsorbers can significantly speed laboratory-scale isolation of antibodies and other Fc fragment-expressing proteins. Appropriate analysis and quantification methods can further accelerate protein processing, allowing isolation/characterization to be completed in one workday, instead of 20+ work hours.

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Immunology and Infection

Detection of True IgE-expressing Mouse B Lineage Cells
Michael P. Gallagher *1, Akritee Shrestha *1, Jennifer M. Magee 1, Duane R. Wesemann 1
1Department of Medicine, Division of Rheumatology, Immunology and Allergy, Brigham and Women's Hospital and Harvard Medical School

In vitro analysis of class switch recombination in mice is challenging due to cytophilic IgE molecules bound to Fc receptors on the surface of B cells. We describe a method for IgE detection using trypsin-mediated cleavage of surface-bound IgE prior to fixation and permeabilization for cytoplasmic fluorescence staining.

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Chemistry

Preparation and Characterization of SDF-1α-Chitosan-Dextran Sulfate Nanoparticles
Andrew R. Bader 1, Tina Li 1, Weiping Wang 2, Daniel S. Kohane 2, Joseph Loscalzo 1, Ying-Yi Zhang 1
1Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, 2Laboratory for Biomaterials and Drug Delivery, Department of Anesthesiology, Division of Critical Care, Boston Children's Hospital

The objective of this protocol is to incorporate SDF-1α, a stem cell homing factor, into dextran sulfate-chitosan nanoparticles. The resultant particles are measured for their size and zeta potential, as well as the content, activity, and in vitro release rate of SDF-1α from the nanoparticles.

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Neuroscience

Optimized Management of Endovascular Treatment for Acute Ischemic Stroke
Katharina Schregel 1,2, Daniel Behme 1, Ioannis Tsogkas 1, Michael Knauth 1, Ilko Maier 3, André Karch 4, Rafael Mikolajczyk 4,5, Mathias Bähr 3, Jörn Schäper 6, José Hinz 6, Jan Liman 3, Marios-Nikos Psychogios 1
1Institute of Neuroradiology, University Medical Center Goettingen, 2Department of Radiology, Brigham and Women's Hospital and Harvard Medical School, 3Department of Neurology, University Medical Center Goettingen, 4Department of Epidemiology, Helmholtz Center for Infection Research, 5Institute of Medical Epidemiology, Biostatistics and Informatics, Martin-Luther-University Halle-Wittenberg, 6Department of Anesthesiology, University Medical Center Goettingen

The outcome of patients with acute ischemic stroke depends on swift restoration of cerebral blood flow. This protocol aims at optimizing the management of such patients by minimizing peri-procedural timings and rendering the time from hospital admission to reperfusion as short as possible.

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Immunology and Infection

Fecal (micro) RNA Isolation
Fyonn H. Dhang 1,2, Howard L. Weiner 1,2, Shirong Liu 1,2
1Ann Romney Center for Neurologic Diseases, Department of Neurology, Partners Multiple Sclerosis Center, Brigham and Women's Hospital and Harvard Medical School, 2Evergrande Center for Immunologic Diseases, Harvard Medical School and Brigham and Women's Hospital

This protocol isolates high quality total RNA from fecal samples of animal and human subjects. A commercial miRNA isolation kit is used with significant adaption to isolate pure RNA with optimized quantity and quality. The RNA isolates are good for most downstream RNA assays such as sequencing, micro-array, and RT-PCR.

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