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Masaryk University

9 ARTICLES PUBLISHED IN JoVE

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JoVE Journal

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products
Brad L. Upham 1, Iva Sovadinová 2, Pavel Babica 2
1Department of Pediatrics & Human Development, Institute for Integrative Toxicology, Michigan State University, 2RECETOX — Research Centre for Toxic Compounds in the Environment, Faculty of Science, Masaryk University

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

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Biochemistry

Spectrophotometric Determination of Phycobiliprotein Content in Cyanobacterium Synechocystis
Tomáš Zavřel 1, Dominik Chmelík 1,2, Maria A. Sinetova 3, Jan Červený 1
1Department of Adaptive Biotechnologies, Global Change Research Institute, Czech Academy of Sciences, 2Department of Plant Physiology, Faculty of Science, Masaryk University, 3Laboratory of Intracellular Regulation, Institute of Plant Physiology, Russian Academy of Sciences

Here, we present a protocol to quantitatively determine phycobiliprotein content in the cyanobacterium Synechocystis using a spectrophotometric method. The extraction procedure was also successfully applied to other cyanobacteria and algae strains; however, due to variations in pigment absorption spectra, it is necessary to test the spectrophotometric equations for each strain individually.

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Developmental Biology

Human Egg Maturity Assessment and Its Clinical Application
Zuzana Holubcová 1,2, Drahomíra Kyjovská 1, Martina Martonová 1, Darja Páralová 1, Tereza Klenková 1, Soňa Kloudová 1
1Reprofit International, Clinic of Reproductive Medicine, 2Department of Histology and Embryology, Faculty of Medicine, Masaryk University

We provide an outline of the clinical protocol for non-invasive assessment of human egg maturity using polarized light microscopy.

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Biochemistry

Cryo-EM and Single-Particle Analysis with Scipion
A. Jiménez-Moreno 1, L. del Caño 1, M. Martínez 1, E. Ramírez-Aportela 1, A. Cuervo 1, R. Melero 1, R. Sánchez-García 1, D. Strelak 1,2,3, E. Fernández-Giménez 1, F.P. de Isidro-Gómez 1, D. Herreros 1, P. Conesa 1, Y. Fonseca 1, D. Maluenda 1, J. Jiménez de la Morena 1, J.R. Macías 1, P. Losana 1, R. Marabini 1, J.M. Carazo 1, C.O.S. Sorzano 1,4
1Centro Nacional de Biotecnología, Campus Universidad Autónoma de Madrid, 2Faculty of Informatics, Masaryk University, 3Institute of Computer Science, Masaryk University, 4Campus Urbanización Montepríncipe, Universidad San Pablo CEU

Single-particle analysis in cryo-electron microscopy is one of the main techniques used to determine the structure of biological ensembles at high resolution. Scipion provides the tools to create the whole pipeline to process the information acquired by the microscope and achieve a 3D reconstruction of the biological specimen.

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Biochemistry

Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography
Jana Moravcová 1, Matyáš Pinkas 1, Radka Holbová 1, Jiří Nováček 1
1Central European Institute of Technology, Masaryk University

We present a protocol for lamella preparation of plunge frozen biological specimens by cryo-focused ion beam micromachining for high-resolution structural studies of macromolecules in situ with cryo-electron tomography. The presented protocol provides guidelines for the preparation of high-quality lamellae with high reproducibility for structural characterization of macromolecules inside the Saccharomyces cerevisiae.

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Developmental Biology

Rapid Isolation of Single Cells from Mouse and Human Teeth
Jan Krivanek 1, Josef Lavicky 1, Thibault Bouderlique 2, Igor Adameyko 2,3
1Department of Histology and Embryology, Faculty of Medicine, Masaryk University, 2Department of Molecular Neuroimmunology, Centre for Brain Research, Medical University of Vienna, 3Department of Physiology and Pharmacology, Karolinska Institute

The current protocol presents a fast, efficient, and gentle method for isolating single cells suitable for single-cell RNA-seq analysis from a continuously growing mouse incisor, mouse molar, and human teeth.

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Biology

Measurement of Liver Stiffness Using Atomic Force Microscopy Coupled with Polarization Microscopy
Srikant Ojha 1, Jan Pribyl 2, Simon Klimovic 2,3, Daniel Hadraba 4, Marketa Jirouskova 1, Martin Gregor 1
1Laboratory of Integrative Biology, Institute of Molecular Genetics of the Czech Academy of Sciences, 2CEITEC MU, Masaryk University, 3Department of Biochemistry, Faculty of Science, Masaryk University, 4Laboratory of Biomathematics, Czech-BioImaging IPHYS, Institute of Physiology of the Czech Academy of Sciences

We present a protocol to measure the elastic moduli of collagen-rich areas in normal and diseased liver using atomic force microscopy. The simultaneous use of polarization microscopy provides high spatial precision for localizing collagen-rich areas in the liver sections.

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Biology

Non-invasive Assay for Chlorophyll Biosynthesis Kinetics Determination during Early Stages of Arabidopsis De-etiolation
Veronika Balakhonova *1,2, Nadiia Pushkarova *1,3, Jan Skalak 1,2, Tereza Dobisova 1,2,4, Zuzana Benedikty 5, Klara Panzarova 5, Martin Trtilek 5, Jan Hejátko 1,2
1CEITEC - Central European Institute of Technology, Masaryk University, 2National Centre for Biomolecular Research, Faculty of Science, Masaryk University, 3Institute of Food Biotechnology and Genomics, National Academy of Sciences of Ukraine, 4Labdeers s.r.o, 5Photon Systems Instruments, Prumyslova

Here, we describe an advanced tool designed for chlorophyll biosynthesis monitoring during the early stages of Arabidopsis seedling de-etiolation. The novel methodology provides non-invasive real-time chlorophyll fluorescence imaging at high spatial and temporal resolution.

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Biology

Hairy Root Transformation and Regeneration in Arabidopsis thaliana and Brassica napus
Veronika Jedličková 1, Marie Štefková 1, Marek Sedláček 1, Klára Panzarová 2, Helene S. Robert 1
1Hormonal Crosstalk in Plant Development, Mendel Center for Plant Genomics and Proteomics, CEITEC MU-Central European Institute of Technology, Masaryk University, 2PSI - Photon Systems Instruments

The protocol describes hairy root induction using Arabidopsis primary inflorescence stems and Brassica napus hypocotyls. The hairy roots can be cultured and used as explants to regenerate transgenic plants.

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