Technical University Munich 5 articles published in JoVE Biology Isolation of Nuclei from Flash-Frozen Liver Tissue for Single-Cell Multiomics Mateusz Strzelecki1,4, Kelvin Yin1, Carlos Talavera-López2,3, Celia P. Martinez-Jimenez1,4 1Helmholtz Pioneer Campus (HPC), Helmholtz Munich, 2Institute of Computational Biology, Computational Health Department, Helmholtz Munich, 3Division of Infectious Diseases and Tropical Medicine, Ludwig-Maximillian-Universität Klinikum, 4TUM School of Medicine, Technical University of Munich Here, we present a protocol for isolating nuclei from flash-frozen, archived liver tissues for single-nucleus RNA-seq, ATAC-seq, and joint multiomics (RNA-seq and ATAC-seq). Cancer Research Syngeneic Mouse Orthotopic Allografts to Model Pancreatic Cancer Constantin Schmitt1,2,3, Dieter Saur1,2,3,4, Stefanie Bärthel*1,2,3, Chiara Falcomatà*1,2,3 1Division of Translational Cancer Research, German Cancer Research Center (DKFZ) and German Cancer Consortium (DKTK), 2Translational Cancer Research and Institute of Experimental Cancer Therapy, Klinikum rechts der Isar, School of Medicine, Technical University of Munich, 3Center for Translational Cancer Research (TranslaTUM), School of Medicine, Technical University of Munich, 4Department of Internal Medicine II, Klinikum rechts der Isar, Technical University of Munich Syngeneic mouse orthotopic allografts of pancreatic ductal adenocarcinoma (PDAC) recapitulate the biology, phenotypes, and therapeutic responses of disease subtypes. Owing to their fast, reproducible tumor progression, they are widely used in preclinical studies. Here, we show common practices to generate these models, injecting syngeneic murine PDAC cultures into the pancreas. Neuroscience Microfluidics-Assisted Selective Depolarization of Axonal Mitochondria Simone Wanderoy*1,2, Alina Rühmkorf*1,2, Angelika B. Harbauer2,3,4 1TUM Medical Graduate Center, Technical University of Munich, 2Max Planck Institute of Neurobiology, 3TUM School of Medicine, Institute of Neuronal Cell Biology, Technical University of Munich, 4Munich Cluster for Systems Neurology The present protocol describes the seeding and staining of neuronal mitochondria in microfluidic chambers. The fluidic pressure gradient in these chambers allows for the selective treatment of mitochondria in axons to analyze their properties in response to pharmacological challenges without affecting the cell body compartment. Medicine Combining 3D-Printing and Electrospinning to Manufacture Biomimetic Heart Valve Leaflets Benedikt Freystetter1, Maximilian Grab1,2, Linda Grefen1, Lara Bischof1, Lorenz Isert3, Petra Mela2, Deon Bezuidenhout4, Christian Hagl1,5, Nikolaus Thierfelder1 1Department of Cardiac Surgery, Ludwig Maximilians University Munich, 2Chair of Medical Materials and Implants, Technical University Munich, 3Faculty for Chemistry and Pharmacy, Ludwig Maximilians University Munich, 4Cardiovascular Research Unit, University of Cape Town, 5DZHK (German Centre for Cardiovascular Research), partner site Munich Heart Alliance The presented method offers an innovative way for engineering biomimetic fiber structures in three-dimensional (3D) scaffolds (e.g., heart valve leaflets). 3D-printed, conductive geometries were used to determine shape and dimensions. Fiber orientation and characteristics were individually adjustable for each layer. Multiple samples could be manufactured in one setup. Biochemistry Mass-Sensitive Particle Tracking to Characterize Membrane-Associated Macromolecule Dynamics Frederik Steiert1,2, Tamara Heermann1, Nikolas Hundt3, Petra Schwille1 1Department of Cellular and Molecular Biophysics, Max Planck Institute of Biochemistry, 2Department of Physics, Technical University Munich, 3Department of Cellular Physiology, Biomedical Center (BMC), Ludwig-Maximilians-Universität München This protocol describes an iSCAT-based image processing and single-particle tracking approach that enables the simultaneous investigation of the molecular mass and the diffusive behavior of macromolecules interacting with lipid membranes. Step-by-step instructions for sample preparation, mass-to-contrast conversion, movie acquisition, and post-processing are provided alongside directions to prevent potential pitfalls.
