Universidade do Porto 9 articles published in JoVE Biology Flow Cytometry Analysis of Murine Bone Marrow Hematopoietic Stem and Progenitor Cells and Stromal Niche Cells Laura Mosteo1, Joana Reis1, Lídia Rocha1, Marta Lopes1, Delfim Duarte1,2,3,4 1Instituto de Investigação e Inovação em Saúde (i3S), Universidade do Porto, 2Department of Onco-Hematology, Instituto Português de Oncologia (IPO)-Porto, 3Unit of Biochemistry, Department of Biomedicine, Faculdade de Medicina da Universidade do Porto, 4Porto Comprehensive Cancer Center (P.CCC) Here, we describe a simple protocol for the isolation and staining of murine bone marrow cells to phenotype hemopoietic stem and progenitor cells along with the supporting niche endothelial and mesenchymal stem cells. A method to enrich cells located in endosteal and central bone marrow areas is also included. Biology Isolation and Characterization of Cyanobacterial Extracellular Vesicles Steven J. Biller*1, María del Carmen Muñoz-Marín*2, Steeve Lima3,4,5, Jorge Matinha-Cardoso3,4, Paula Tamagnini3,4,6, Paulo Oliveira3,4,6 1Department of Biological Sciences, Wellesley College, 2Departamento de Bioquímica y Biología Molecular, Campus de Excelencia Internacional, Agroalimentario, Universidad de Córdoba, 3i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, 4IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, 5MCbiology Doctoral Program, ICBAS - Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, 6Departamento de Biologia, Faculdade de Ciências, Universidade do Porto The present protocol providesdetailed descriptions for isolation, concentration,and characterization of extracellular vesicles from cyanobacterial cultures. Approaches for purifying vesicles from cultures at different scales, trade-offs among methodologies, and considerations for working with field samples are also discussed. Medicine Measurement of Tissue Non-Heme Iron Content using a Bathophenanthroline-Based Colorimetric Assay Tiago L. Duarte*1,2, João V. Neves*1,3,4 1i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, 2Basic and Clinical Research on Iron Biology, IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, 3Iron and Innate Immunity, IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, 4ICBAS - Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto Here, a protocol for the measurement of the non-heme iron content in animal tissues is provided, using a simple, well-established colorimetric assay that can be easily implemented in most laboratories. Medicine Studying Left Ventricular Reverse Remodeling by Aortic Debanding in Rodents Patricia Goncalves-Rodrigues*1, Daniela Miranda-Silva*1, Adelino F. Leite-Moreira1, Inês Falcão-Pires1 1Unidade de Investigação Cardiovascular, Departamento de Cirurgia e Fisiologia, Faculdade de Medicina, Universidade do Porto Here we describe a step-by-step protocol of surgical aorta debanding in the well-established mice model of aortic-constriction. This procedure not only allows studying the mechanisms underlying the left ventricular reverse remodeling and regression of hypertrophy but also to test novel therapeutic options that might accelerate myocardial recovery. Developmental Biology Fluorescent Immunolocalization of Arabinogalactan Proteins and Pectins in the Cell Wall of Plant Tissues Mário Costa1,2, Ana Marta Pereira3, Sílvia Coimbra1,2 1Departamento de Biologia, Faculdade de Ciências da Universidade do Porto, 2Laboratório Associado para a Química Verde, 3Dipartimento di Bioscienze, Universitá degli Studi di Milano This protocol describes in detail how the plant material for immunolocalization of Arabinogalactan proteins and pectins is fixed, embedded in a hydrophilic acrylic resin, sectioned and mounted on glass slides. We show cell wall related epitopes will be detected with specific antibodies. Medicine In Vitro Assessment of Cardiac Function Using Skinned Cardiomyocytes Patrícia Gonçalves-Rodrigues*1, João Almeida-Coelho*1, Alexandre Gonçalves1, Flávio Amorim1, Adelino F. Leite-Moreira1, Ger J.M. Stienen2, Inês Falcão-Pires1 1Unidade de Investigação Cardiovascular, Departamento de Cirurgia e Fisiologia, Faculdade de Medicina, Universidade do Porto, 2Department of Physiology, Kilimanjaro Christian Medical University College This protocol aims to describe step-by-step the technique of extraction and assessment of cardiac function using skinned cardiomyocytes. This methodology allows measurement and acutemodulation of myofilament function using small frozen biopsies that can be collected from different cardiac locations, from mice to men. Biochemistry Looking Outwards: Isolation of Cyanobacterial Released Carbohydrate Polymers and Proteins Carlos Flores1,2,3, Paula Tamagnini1,2,4 1Bioengineering and Synthetic Microbiology Group, Instituto de Investigação e Inovação em Saude, Universidade do Porto, 2Instituto de Biologia Celular e Molecular, Universidade do Porto, 3Instituto de Ciências Biomédicas Abel Salazar, 4Departamento de Biologia, Faculdade de Ciências, Universidade do Porto Here, protocols for the isolation of cyanobacterial released carbohydrate polymers and isolation of their exoproteomes are described. Both procedures embody key steps to obtain polymers or proteins with high purity degrees that can be used for further analysis or applications. They can also be easily adapted according to specific user needs. Bioengineering Interfacing Microfluidics with Microelectrode Arrays for Studying Neuronal Communication and Axonal Signal Propagation Cátia D.F. Lopes1,2, José C. Mateus1,2,3, Paulo Aguiar1,2 1i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, 2INEB - Instituto de Engenharia Biomédica, Universidade do Porto, 3ICBAS - Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto This protocol aims to demonstrate how to combine in vitro microelectrode arrays with microfluidic devices for studying action potential transmission in neuronal cultures. Data analysis, namely the detection and characterization of propagating action potentials, is performed using a new advanced, yet user-friendly and freely available, computational tool. Immunology and Infection Normal and Malignant Muscle Cell Transplantation into Immune Compromised Adult Zebrafish Inês M. Tenente*1,2,3, Qin Tang*1,2, John C. Moore1,2, David M. Langenau1,2 1Molecular Pathology, Cancer Center and Center for Regenerative Medicine, Massachusetts General Hospital, 2Harvard Stem Cell Institute, 3GABBA - Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto Here, we present a protocol for cell transplantation of zebrafish skeletal muscle and embryonal rhabdomyosarcoma (ERMS) into adult immune compromised rag2E450fs homozygous mutant zebrafish. This protocol allows for the efficient analysis of regeneration and malignant transformation of muscle cells.
