University of South Alabama 6 articles published in JoVE Biology Integrative Toolkit to Analyze Cellular Signals: Forces, Motion, Morphology, and Fluorescence Alyson Nguyen*1, Keith Battle*2,3, Sunita S. Paudel*2,3, Ningyong Xu2, Jessica Bell3, Linn Ayers3, Cassandra Chapman4, Ajay P. Singh5, Srinivas Palanki6, Thomas Rich3,7, Diego F. Alvarez8, Troy Stevens2,3, Dhananjay T. Tambe3,4,7 1Biomedical Sciences, Pat Capps Covey College of Allied Health Professions, University of South Alabama, 2Department of Physiology and Cell Biology, College of Medicine, University of South Alabama, 3Center for Lung Biology, College of Medicine, University of South Alabama, 4William B. Burnsed Jr. Mechanical, Aerospace, and Biomedical Engineering Department, College of Engineering, University of South Alabama, 5Mitchell Cancer Institute, College of Medicine, University of South Alabama, 6Department of Chemical and Biomedical Engineering, West Virginia University, 7Department of Pharmacology, College of Medicine, University of South Alabama, 8Department of Physiology & Pharmacology, College of Osteopathic Medicine, Sam Houston State University The Integrative Toolkit to Analyze Cellular Signals (iTACS) platform automates the process of simultaneously measuring a wide variety of chemical and mechanical signals in adherent cells. iTACS is designed to facilitate community-driven development and enable researchers to use all platform features regardless of their educational background. Biology Measurement of 3-Dimensional cAMP Distributions in Living Cells using 4-Dimensional (x, y, z, and λ) Hyperspectral FRET Imaging and Analysis Naga S. Annamdevula1,2, Rachel Sweat3, Hayden Gunn1, John R. Griswold3, Andrea L. Britain1,2, Thomas C. Rich1,2, Silas J. Leavesley1,2,3 1Department of Pharmacology, University of South Alabama, 2Center for Lung Biology, University of South Alabama, 3Department of Chemical and Biomolecular Engineering, University of South Alabama Due to inherent low signal-to-noise ratio (SNR) of Fӧrster resonance energy transfer (FRET) based sensors, measurement of cAMP signals has been challenging, especially in three spatial dimensions. Here, we describe a hyperspectral FRET imaging and analysis methodology that allows measurement of cAMP distribution in three spatial dimensions. Engineering Excitation-Scanning Hyperspectral Imaging Microscopy to Efficiently Discriminate Fluorescence Signals Joshua Deal1,2,3, Andrea Britain2,3, Thomas Rich2,3, Silas Leavesley1,2,3 1Department of Chemical and Biomolecular Engineering, University of South Alabama, 2Center for Lung Biology, University of South Alabama, 3Department of Pharmacology, University of South Alabama Spectral imaging has become a reliable solution for identification and separation of multiple fluorescence signals in a single sample and can readily distinguish signals of interest from background or autofluorescence. Excitation-scanning hyperspectral imaging improves on this technique by decreasing the necessary image acquisition time while simultaneously increasing the signal-to-noise ratio. Immunology and Infection Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa Ron Balczon1,4, Michael Francis2,4, Silas Leavesley3,4, Troy Stevens2,4 1Department of Biochemistry and Molecular Biology, University of South Alabama, 2Department of Physiology and Cell Biology, University of South Alabama, 3Department of Chemical and Biomolecular Engineering, University of South Alabama, 4Center for Lung Biology, University of South Alabama Simple methods are described for demonstrating the production of cytotoxic amyloids following infection of pulmonary endothelium by Pseudomonas aeruginosa. Developmental Biology Reprogramming Primary Amniotic Fluid and Membrane Cells to Pluripotency in Xeno-free Conditions Jaroslav Slamecka1, Javier Laurini2, Troy Shirley1, Simon Philipp Hoerstrup3, Benedikt Weber3,4,5, Laurie Owen1, Steven McClellan1 1Mitchell Cancer Institute, University of South Alabama, 2College of Medicine, University of South Alabama, 3Institute for Regenerative Medicine, University of Zurich, 4Department of Dermatology, University Hospital Zurich, 5Center for Applied Biotechnology and Molecular Medicine (CABMM), University of Zurich - Irchel Campus This protocol describes the reprogramming of primary amniotic fluid and membrane mesenchymal stem cells into induced pluripotent stem cells using a non-integrating episomal approach in fully chemically defined conditions. Procedures of extraction, culture, reprogramming, and characterization of the resulting induced pluripotent stem cells by stringent methods are detailed. Biology Automated Analysis of Dynamic Ca2+ Signals in Image Sequences Michael Francis1, Josh Waldrup2, Xun Qian2, Mark S. Taylor2 1Department of Pharmacology, University of South Alabama, 2Department of Physiology, University of South Alabama Here a novel region of interest analysis protocol based on sorting best-fit ellipses assigned to regions of positive signal within two-dimensional time lapse image sequences is demonstrated. This algorithm may enable investigators to comprehensively analyze physiological Ca2+ signals with minimal user input and bias.
