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Boston Children’s Hospital

7 ARTICLES PUBLISHED IN JoVE

Bioengineering

Manufacture of Concentrated, Lipid-based Oxygen Microbubble Emulsions by High Shear Homogenization and Serial Concentration

Lindsay M. Thomson¹, Brian D. Polizzotti¹, Frances X. McGowan², John N. Kheir¹

¹Department of Cardiology, Boston Children's Hospital, Harvard Medical School, ²Department of Anesthesiology and Critical Care, Children's Hospital of Philadelphia, University of Pennsylvania

We describe methods for the manufacture of large volumes of lipid-based oxygen microbubbles (LOMs) designed for intravenous oxygen delivery using high-shear homogenization and serial concentration.

Immunology and Infection

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

Cheryn J. Couter¹, Neeraj K. Surana¹

¹Division of Infectious Diseases, Department of Medicine, Boston Children’s Hospital

There is growing interest in the quantitative characterization of intestinal lymphocytes owing to increasing recognition that these cells play a critical role in a variety of intestinal and systemic diseases. In this protocol, we describe how to isolate single cell populations from different small-intestinal compartments for subsequent flow cytometric characterization.

Biology

Rodent Working Heart Model for the Study of Myocardial Performance and Oxygen Consumption

Elizabeth S. DeWitt^*1, Katherine J. Black^*1, John N. Kheir¹

¹Department of Cardiology, Boston Children’s Hospital

Isolated working heart models can be used to measure the effect of loading conditions, heart rate, and medications on myocardial performance and oxygen consumption. We describe methods for preparation of a rodent left heart working model that permits study of systolic and diastolic performance and oxygen consumption under various conditions.

Developmental Biology

Generation of Parabiotic Zebrafish Embryos by Surgical Fusion of Developing Blastulae

Elliott J. Hagedorn^1,2, Jennifer L. Cillis³, Caitlyn R. Curley³, Taylor C. Patch³, Brian Li^1,2, Bradley W. Blaser^1,2,7, Raquel Riquelme^1,2, Leonard I. Zon^1,2,4,5,6, Dhvanit I. Shah^1,2,3,4,5

¹Division of Hematology/Oncology, Boston Children’s Hospital, ²Harvard Medical School, ³Division of Hematology, Department of Medicine, Brigham and Women’s Hospital, ⁴Harvard Stem Cell Institute, ⁵Broad Institute of Massachusetts Institute of Technology, ⁶Howard Hughes Medical Institute, ⁷Division of Hematologic Malignancies, Dana-Farber Cancer Institute

This protocol provides step-by-step instruction on how to generate parabiotic zebrafish embryos of different genetic backgrounds. When combined with the unparalleled imaging capabilities of the zebrafish embryo, this method provides a uniquely powerful means to investigate cell-autonomous versus non-cell-autonomous functions for candidate genes of interest.

Neuroscience

Measuring and Altering Mating Drive in Male Drosophila melanogaster

Christine L. Boutros^*1, Lauren E. Miner^*1, Ofer Mazor^2,3, Stephen X. Zhang³

¹F.M. Kirby Neurobiology Center, Boston Children’s Hospital, ²Harvard NeuroDiscovery Center, Harvard Medical School, ³Department of Neurobiology, Harvard Medical School

This article describes a behavioral assay that uses male mating drive in Drosophila melanogaster to study motivation. Using this method, researchers can utilize advanced fly neurogenetic techniques to uncover the genetic, molecular, and cellular mechanisms that underlie this motivation.

JoVE Journal

Analysis of Hematopoietic Stem Progenitor Cell Metabolism

Giorgia Scapin^1,2,3, Marie C. Goulard^1,2,3, Priyanka R. Dharampuriya^1,2,3, Jennifer L. Cillis^1,2,3, Dhvanit I. Shah^1,2,3

¹Nationwide Children's Hospital, ²The Ohio State University College of Medicine, ³The Ohio State University Comprehensive Cancer Center

Hematopoietic stem progenitor cells (HSPCs) transition from a quiescent state to a differentiation state due to their metabolic plasticity during blood formation. Here, we present an optimized method for measuring mitochondrial respiration and glycolysis of HSPCs.

Genetics

Monochrome Multiplex Quantitative PCR Telomere Length Measurement

Noelle A. Martin^*1, Lauren W. Y. McLester-Davis^*2,3,4, Trevor R. Roy¹, Madeline G. Magruder⁵, Waylon J. Hastings¹, Stacy S. Drury⁶

¹School of Medicine, Tulane University, ²Native American Center for Health Professions, University of Wisconsin-Madison, ³Department of Medicine, University of Wisconsin-Madison, ⁴Department of Biochemistry, University of Wisconsin-Madison, ⁵School of Science and Engineering, Tulane University, ⁶Department of Psychiatry and Behavioral Sciences, Boston Children’s Hospital

Here we present a protocol for the measurement of relative telomere length (TL) using the monochrome multiplex quantitative polymerase chain reaction (MMqPCR) assay. The MMqPCR assay is a repeatable, efficient, and cost-effective method for measuring TL from human DNA in population-based studies.