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Universidade do Porto

11 ARTICLES PUBLISHED IN JoVE

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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. Moore 1,2, David M. Langenau 1,2
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.

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Biology

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry
Sandrine Schmutz 1, Mariana Valente 2,3,4,5, Ana Cumano 2, Sophie Novault 1
1Flow Cytometry Core Facility, Center for Translational Research-Technical Core, Institut Pasteur, 2Unit for Lymphopoiesis, Immunology Department, INSERM U1223, University Paris Diderot, Sorbonne Paris Cité, Cellule Pasteur, Institut Pasteur, 3Stem-Cell Microenvironments in Repair/Regeneration Team, Instituto de Investigação e Inovação em Saúde (i3s), INEB - Instituto de Engenharia Biomédica, 4ICBAS - Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, 5Stem Cells and Regenerative Medicine Team, UMRS 1166, ICAN - Institute of Cardiometabolism And Nutrition, UPMC - Université Pierre et Marie Curie - Paris 6, INSERM

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

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Neuroscience

Modified Roller Tube Method for Precisely Localized and Repetitive Intermittent Imaging During Long-term Culture of Brain Slices in an Enclosed System
Benjamin B. Fixman *1, Isaac W. Babcock *1, Laurie S. Minamide *1, Alisa E. Shaw 1, Marina I. Oliveira da Silva 1,2, Avery M. Runyan 1, Michael T. Maloney 1,3, Jeffrey J. Field 1, James R. Bamburg 1
1Department of Biochemistry and Molecular Biology and Molecular, Cellular and Integrated Neuroscience Program, Colorado State University, 2IBMC-Instituto de Biologia Molecular e Celular, i3S-Instituto de Investigaçãoe Inovação em Saúde, ICBAS, Universidade do Porto, 3Denali Therapeutics

Presented here is a modified roller tube method for culturing and intermittent high-resolution imaging of rodent brain slices over many weeks with precise repositioning on photoetched coverslips. Neuronal viability and slice morphology are well maintained. Applications of this fully enclosed system using viruses for cell-type specific expression are provided.

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Bioengineering

Comparable Decellularization of Fetal and Adult Cardiac Tissue Explants as 3D-like Platforms for In Vitro Studies
Ana C. Silva 1,2,3,4, Maria J. Oliveira 1,2,5, Todd C McDevitt 4,6, Mário A. Barbosa 1,2,3, Diana S. Nascimento 1,2, Perpétua Pinto-do-Ó 1,2,3
1i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, 2INEB - Instituto de Engenharia Biomédica, Universidade do Porto, 3Instituto de Ciências Biomédicas Abel Salazar (ICBAS), Universidade do Porto, 4Gladstone Institute of Cardiovascular Disease, 5Faculty of Medicine, University of Porto, 6University of California San Francisco

The cardiac extracellular matrix (ECM) is a complex network of molecules that orchestrate key processes in tissues and organs while enduring physiological remodeling throughout life. Standardized decellularization of fetal and adult hearts permits comparative experimental studies of both tissues in a 3D context by capturing native architecture and biomechanical properties.

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Bioengineering

Interfacing Microfluidics with Microelectrode Arrays for Studying Neuronal Communication and Axonal Signal Propagation
Cátia D.F. Lopes 1,2, José C. Mateus 1,2,3, Paulo Aguiar 1,2
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.

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JoVE Journal

Looking Outwards: Isolation of Cyanobacterial Released Carbohydrate Polymers and Proteins
Carlos Flores 1,2,3, Paula Tamagnini 1,2,4
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.

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Medicine

Studying Left Ventricular Reverse Remodeling by Aortic Debanding in Rodents
Patricia Goncalves-Rodrigues *1, Daniela Miranda-Silva *1, Adelino F. Leite-Moreira 1, Inês Falcão-Pires 1
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.

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Medicine

In Vitro Assessment of Cardiac Function Using Skinned Cardiomyocytes
Patrícia Gonçalves-Rodrigues *1, João Almeida-Coelho *1, Alexandre Gonçalves 1, Flávio Amorim 1, Adelino F. Leite-Moreira 1, Ger J.M. Stienen 2, Inês Falcão-Pires 1
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.

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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.

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Biology

Isolation and Characterization of Cyanobacterial Extracellular Vesicles
Steven J. Biller *1, María del Carmen Muñoz-Marín *2, Steeve Lima 3,4,5, Jorge Matinha-Cardoso 3,4, Paula Tamagnini 3,4,6, Paulo Oliveira 3,4,6
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.

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JoVE Core

Flow Cytometry Analysis of Murine Bone Marrow Hematopoietic Stem and Progenitor Cells and Stromal Niche Cells
Laura Mosteo 1, Joana Reis 1, Lídia Rocha 1, Marta Lopes 1, Delfim Duarte 1,2,3,4
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.

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