University of Arizona College of Medicine - Phoenix View Institution's Website 6 articles published in JoVE Biochemistry A Reporter Based Cellular Assay for Monitoring Splicing Efficiency Jason Wong1, William Martelly1, Shalini Sharma1 1Department of Basic Medical Sciences, College of Medicine-Phoenix, University of Arizona This protocol describes a minigene reporter assay to monitor the impact of 5´-splice site mutations on splicing and develops suppressor U1 snRNA for the rescue of mutation-induced splicing inhibition. The reporter and suppressor U1 snRNA constructs are expressed in HeLa cells, and splicing is analyzed by primer extension or RT-PCR. Developmental Biology Pan-myeloid Differentiation of Human Cord Blood Derived CD34+ Hematopoietic Stem and Progenitor Cells Aditi Bapat1, Nakia Keita1, Shalini Sharma1 1Department of Basic Medical Sciences, College of Medicine-Phoenix, University of Arizona Here, we present a protocol for immunophenotypic characterization and cytokine induced differentiation of cord blood derived CD34+ hematopoietic stem and progenitor cells to the four myeloid lineages. The applications of this protocol include investigations on the effect of myeloid disease mutations or small molecules on myeloid differentiation of the CD34+ cells. Neuroscience Simultaneous Cryosectioning of Multiple Rodent Brains Tabitha R.F. Green1,2,3, J. Bryce Ortiz1, Jordan L. Harrison5, Jonathan Lifshitz1,3,4, Rachel K. Rowe1,3,4 1Department of Child Health, University of Arizona College of Medicine, 2Department of Biological Sciences, University of Bath, 3BARROW Neurological Institute at Phoenix Children's Hospital, 4Phoenix Veteran Affairs Healthcare System, 5Department of Basic Medical Sciences, University of Arizona College of Medicine Here, we present a protocol to freeze and section brain tissue from multiple animals as a timesaving alternative to processing single brains. This reduces staining variability during immunohistochemistry and reduces time cryosectioning and imaging. Neuroscience A Simplified Method for Ultra-Low Density, Long-Term Primary Hippocampal Neuron Culture Zhongming Lu*1,2, Mariel Piechowicz*1, Shenfeng Qiu1 1Department of Basic Medical Sciences, University of Arizona College of Medicine-Phoenix, 2Jiangsu Provincial Center for Disease Control and Prevention Low density cultures of primary hippocampal neurons usually require glia feeder layer to supply neurotrophic factors and sustain longevity. We describe here a simplified method to culture ultra-low density neurons on glass coverslips in the presence of a high density neuronal feeder layer, which facilitates investigation of specific neuronal-autonomous mechanisms. Neuroscience Primer for Immunohistochemistry on Cryosectioned Rat Brain Tissue: Example Staining for Microglia and Neurons Megan N. Evilsizor1,2, Helen F. Ray-Jones1,3, Jonathan Lifshitz1,2,4,5, Jenna Ziebell1,2 1Department of Child Health, University of Arizona College of Medicine - Phoenix, 2 This introductory level protocol describes the reagents, equipment, and techniques required to complete immunohistochemical staining of rodent brains, using markers for microglia and neuronal elements as an example. Biology Culturing and Applications of Rotating Wall Vessel Bioreactor Derived 3D Epithelial Cell Models Andrea L. Radtke1, Melissa M. Herbst-Kralovetz1 1Basic Medical Sciences, University of Arizona College of Medicine - Phoenix A rotating cell culture system that allows epithelial cells to grow under physiological conditions resulting in 3-D cellular aggregate formation is described. The aggregates generated display in vivo-like characteristics not observed in conventional culture models and serve as a more accurate organotypic model system for a multitude of scientific investigations.