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8 ARTICLES PUBLISHED IN JoVE

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Biology

Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents
Niels Voigt *1,2, Xiao-Bo Zhou *2, Dobromir Dobrev 1,2
1Institute of Pharmacology, University of Duisburg-Essen , 2Division of Experimental Cardiology, University of Heidelberg

We describe the isolation of human atrial myocytes which can be used for intracellular Ca2+ measurements in combination with electrophysiological patch-clamp studies.

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Medicine

Coculture System with an Organotypic Brain Slice and 3D Spheroid of Carcinoma Cells
Han-Ning Chuang 1, Raphaela Lohaus 1, Uwe-Karsten Hanisch 2, Claudia Binder 1, Faramarz Dehghani *3, Tobias Pukrop *1
1Department of Hematology and Oncology, University of Göttingen, 2Institute of Neuropathology, University of Göttingen, 3Institute of Anatomy and Cellbiology, University of Halle

The organotypic brain slice coculture with carcinoma cells enables visualizing morphological changes by fluorescence as well as bright field (video) microscopy during the process of carcinoma cell invasion of brain tissue. This model system also allows for cell exchange and replenishment approaches and offers a wide variety of manipulations and analyses.

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Immunology and Infection

Isolation of Human Monocytes by Double Gradient Centrifugation and Their Differentiation to Macrophages in Teflon-coated Cell Culture Bags
Kerstin Menck 1, Daniel Behme 1, Mathias Pantke 1, Norbert Reiling 2, Claudia Binder 1, Tobias Pukrop 1, Florian Klemm 1
1Department of Hematology and Oncology, University Medical Center Göttingen, 2Microbial Interface Biology Group, Research Center Borstel

We present a simple and efficient protocol for the generation of human macrophages. Buffy coats are processed by double density gradient centrifugation and isolated monocytes are then differentiated to macrophages in Teflon-coated cell culture bags. This maximizes macrophage yields and facilitates cell harvesting for subsequent experiments.

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Neuroscience

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo
Thomas Hassenklöver 1, Ivan Manzini 1
1Institute of Neurophysiology and Cellular Biophysics and Center for Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB), University of Göttingen

We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

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Biology

Isolation and Characterization of Microvesicles from Peripheral Blood
Kerstin Menck 1, Annalen Bleckmann 1, Matthias Schulz 1, Lena Ries 1, Claudia Binder 1
1Department of Hematology/Medical Oncology, University Medical Center Göttingen

Extracellular vesicles present in blood have been suggested as novel biomarkers for various diseases. Here, we present a protocol for the isolation of large plasma membrane-derived microvesicles from peripheral blood samples and their subsequent analysis by conventional flow cytometry and Western Blotting.

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Neuroscience

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster
Clare E. Hancock 1, Florian Bilz 1, André Fiala 1
1Department of Molecular Neurobiology of Behavior, University of Göttingen

Here we present a protocol with which pre- and/or postsynaptic calcium can be visualized in the context of Drosophila learning and memory. In vivo calcium imaging using synaptically localized calcium sensors is combined with a classical olfactory conditioning paradigm such that the synaptic plasticity underlying this type of associative learning may be determined.

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Bioengineering

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes
Fitzwilliam Seibertz 1,2, Martyn Reynolds 3, Niels Voigt 1,2,4
1Institute of Pharmacology and Toxicology, University Medical Center Goettingen, Goettingen, Germany, 2DZHK (German Center for Cardiovascular Research), Partner Site Goettingen, Germany, 3Cairn Research Ltd, Faversham, United Kingdom, 4Cluster of Excellence "Multiscale Bioimaging: from Molecular Machines to Networks of Excitable Cells" (MBExC), University of Goettingen, Germany

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

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Biology

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current
Philipp Tomsits *1,2,3, Dominik Schüttler *1,2,3, Stefan Kääb 1,2, Sebastian Clauss *1,2,3, Niels Voigt *4,5,6
1Department of Medicine I, University Hospital Munich, Campus Großhadern, Ludwig-Maximilians University Munich (LMU), 2Partner Site Munich, Munich Heart Alliance (MHA), DZHK (German Centre for Cardiovascular Research), 3Walter Brendel Center of Experimental Medicine, Ludwig-Maximilians University Munich (LMU), 4Institute of Pharmacology and Toxicology, University Medical Center Göttingen, 5Partner Site Göttingen, DZHK (German Centre for Cardiovascular Research), 6Cluster of Excellence "Multiscale Bioimaging: from Molecular Machines to Networks of Excitable Cells" (MBExC), University of Göttingen

Mouse models allow studying key mechanisms of arrhythmogenesis. For this purpose, high quality cardiomyocytes are necessary to perform patch-clamp measurements. Here, a method to isolate murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, which allows simultaneous measurements of calcium-transients and L-type calcium current, is described.

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