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  • Introduction to Mammalian Cell Culture

    Mammalian cell culture is one of the basic pillars of life sciences. Without the ability to grow cells in the lab, the fast progress in disciplines like cell biology, immunology, or cancer research would be unthinkable. This article gives an overview of mammalian cell culture systems. Mainly, they can be categorized according to their morphology, as well as cell type and organization. Moreover, you can find basic information about the correct growth conditions and what kind of microscope you need to watch your cells.
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  • Five Questions Asked: Prof. Dr. Jacco van Rheenen speaks about the most important considerations when imaging deep into mouse tissue

    When operating a confocal microscope, or when discussing features and parameters of such a device, we inescapably mention the pinhole and its diameter. This short introductory document is meant to explain the significance of the pinhole for those, who did not want to spend too much time to dig into theory and details of confocal microscopy but wanted to have an idea about the effect of the pinhole.
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  • Human NK Cell Development Requires CD56-mediated Motility and Formation of the Developmental Synapse

    While distinct stages of natural killer (NK) cell development have been defined, the molecular interactions that shape human NK cell maturation are poorly understood. Here we define intercellular interactions between developing NK cells and stromal cells which, through contact-dependent mechanisms, promote the generation of mature, functional human NK cells from CD34+ precursors. We show that developing NK cells undergo unique, developmental stage-specific sustained and transient interactions with developmentally supportive stromal cells, and that the relative motility of NK cells increases as they move through development in vitro and ex vivo.
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  • Actin-Dependent Vacuolar Occupancy of the Cell Determines Auxin-Induced Growth Repression

    The cytoskeleton is an early attribute of cellular life, and its main components are composed of conserved proteins. The actin cytoskeleton has a direct impact on the control of cell size in animal cells, but its mechanistic contribution to cellular growth in plants remains largely elusive. Here, we reveal a role of actin in regulating cell size in plants. The actin cytoskeleton shows proximity to vacuoles, and the phytohormone auxin not only controls the organization of actin filaments but also impacts vacuolar morphogenesis in an actin-dependent manner.
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  • Variety of RNAs in Peripheral Blood Cells, Plasma, and Plasma Fractions

    Human peripheral blood contains RNA in cells and in extracellular membrane vesicles, microvesicles and exosomes, as well as in cell-free ribonucleoproteins. Circulating mRNAs and noncoding RNAs, being internalized, possess the ability to modulate vital processes in recipient cells. In this study, with SOLiD sequencing technology, we performed identification, classification, and quantification of RNAs from blood fractions: cells, plasma, plasma vesicles pelleted at 16,000
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  • The Bif-1-Dynamin 2 Membrane Fission Machinery Regulates Atg9-Containing Vesicle Generation at the Rab11-Positive Reservoirs

    Application example of HyVolution Super-Resolution - Atg9 is a multispanning transmembrane protein that is required for autophagosome formation. During autophagy, vesicles containing Atg9 are generated through an unknown mechanism and delivered to the autophagosome formation sites. We have previously reported that Atg9-containing membranes undergo continuous tubulation and fission during nutrient starvation in a manner dependent on the curvature-inducing protein Bif-1/Sh3glb1. Here, we identify Dynamin 2 (DNM2) as a Bif-1-interacting protein that mediates the fission of Atg9-containing membranes during autophagy.
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  • Super-Resolution Optical Microscopy of Lipid Plasma Membrane Dynamics

    Plasma membrane dynamics are an important ruler of cellular activity, particularly through the interaction and diffusion dynamics of membrane-embedded proteins and lipids. FCS (fluorescence correlation spectroscopy) on an optical (confocal) microscope is a popular tool for investigating such dynamics. Unfortunately, its full applicability is constrained by the limited spatial resolution of a conventional optical microscope. The present chapter depicts the combination of optical super-resolution STED (stimulated emission depletion) microscopy with FCS , and why it is an important tool for investigating molecular membrane dynamics in living cells. Compared with conventional FCS , the STED- FCS approach demonstrates an improved possibility to distinguish free from anomalous molecular diffusion, and thus to give new insights into lipid–protein interactions and the traditional lipid ‘raft’ theory.
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  • Practical Guide for Excellent GSDIM Super-Resolution Images

    Do you know that most protists and bacteria lack in one feature that each of our body cell has? Our cells are touch and communicate with one another. They send and receive a variety of signals that coordinate their behavior to act together as a functional multicellular organism. Exploring the way of cellular communication and the ways how the cell surface interacts to organize tissues and body structures is of great interest. Kees Jalink and his team of scientists at the Netherlands Cancer Institute (NKI) in Amsterdam obtained new scientific insights into the molecular architecture of hemidesmosomes, cytoskeletal components, cell surface receptors and vesicular proteins with the help of Ground-State-Depletion (GSD)/ dSTORM microscopy. In this interview, Kees Jalink comments on their developments in imaging chambers, buffer conditions and image analysis to get the perfect super resolution image.
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  • Webinar: Laser Microdissection in Cancer Research – Mutation Analysis Workflow with Pure Cancer Material

    Cancer can affect various organs and is caused by mutations of the DNA. A prerequisite, to explore and understand underlying gene-mutations involved in the development of a definite type of cancer, is the extraction of pure sample material, which is challenging. In this webinar, we will present how to extract 100% pure cancer tissue for DNA analysis with laser microdissection (LMD). Using tissue samples from human kidney cancer patients as an example, this webinar will provide an overview of the practical considerations when preparing a workflow to obtain highly pure material with the LMD microscope for further molecular analysis.
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  • Webinar: Introduction to Fluorescence Microscopy

    In this seminar we will provide an overview about the latest advances in fluorescence microscopy. You will learn how you can use widefield and confocal microscopes to help you understand life’s questions down to tiny details, at high speed and state-of-the-art image quality both in living and fixed samples.
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  • Structural Study of C. elegans

    Application Note for Leica EM ICE, Leica EM AFS2 - Wildtype L4 stage C. elegans (N2 strain) were placed in the 100 μm deep side of Lecithin-coated (see detailed protocol*) type A 3 mm Cu/Au carriers (Leica) with extracellular filler containing 1% (w/v) Agarose type IX and 2% (w/v) Bovine Serum Albumin in bacteria medium (see preparation details**) and sandwiched with the flat side of Lecithin-coated type B 3 mm Cu/Au carriers (Leica). Samples were frozen in a high-pressure freezer (Leica EM ICE).
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  • Freeze-Fracture Replication of Pyramidal Cells

    Application Note for Leica EM HPM100 - Frozen samples (90 μm thick slices frozen by HPM100) were inserted into a double replica table and then fractured into two pieces at –130°C (after insertion of the tissue into BAF 060 the samples should be left in the chamber for 20 min to reach the –130°C).
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  • Adeno-associated Viral Vectors do not Efficiently Target Muscle Satellite Cells

    Adeno-associated viral (AAV) vectors are becoming an important tool for gene therapy of numerous genetic and other disorders. Several recombinant AAV vectors (rAAV) have the ability to transduce striated muscles in a variety of animals following intramuscular and intravascular administration, and have attracted widespread interest for therapy of muscle disorders such as the muscular dystrophies. Here we examined the relative ability of rAAV vectors derived from AAV6 to target myoblasts, myocytes, and myotubes in culture and satellite cells and myofibers in vivo. AAV vectors are able to transduce proliferating myoblasts in culture, albeit with reduced efficiency relative to postmitotic myocytes and myotubes. In contrast, quiescent satellite cells are refractory to transduction in adult mice.
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  • The Actin Cytoskeleton Modulates the Activation of iNKT Cells by Segregating CD1d Nanoclusters on Antigen-Presenting Cells

    The ability of invariant natural killer T (iNKT) cells to recognize endogenous antigens represents a distinct immune recognition strategy, which underscores the constitutive memory phenotype of iNKT cells and their activation during inflammatory conditions. By using superresolution microscopy, we show that CD1d molecules form nanoclusters at the cell surface of APCs, and their size and density are constrained by the actin cytoskeleton.
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  • Highly Selective Fluorescent and Colorimetric Probe for Live-cell Monitoring of Sulphide Based on Bioorthogonal Reaction

    H2S is the third endogenously generated gaseous signaling compound and has also been known to involve a variety of physiological processes. To better understand its physiological and pathological functions, efficient methods for monitoring of H2S are desired. Azide fluorogenic probes are popular because they can take place bioorthogonal reactions. In this work, by employing a fluorescein derivative as the fluorophore and an azide group as the recognition unit, we reported a new probe 5-azidofluorescein for H2S with improved sensitivity and selectivety.
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  • TFG Promotes Organization of Transitional ER and Efficient Collagen Secretion

    Collagen is the most abundant protein in the animal kingdom. It is of fundamental importance during development for cell differentiation and tissue morphogenesis as well as in pathological processes such as fibrosis and cancer cell migration. However, our understanding of the mechanisms of procollagen secretion remains limited. Here, we show that TFG organizes transitional ER (tER) and ER exit sites (ERESs) into larger structures. Depletion of TFG results in dispersion of tER elements that remain associated with individual ER-Golgi intermediate compartments (ERGICs) as largely functional ERESs. We show that TFG is not required for the transport and packaging of small soluble cargoes but is necessary for the export of procollagen from the ER. Our work therefore suggests a key relationship between the structure and function of ERESs and a central role for TFG in optimizing COPII assembly for procollagen export.
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  • The Bimodally Expressed MicroRNA miR‐142 Gates Exit from Pluripotency

    A stem cell's decision to self‐renew or differentiate is thought to critically depend on signaling cues provided by its environment. It is unclear whether stem cells have the intrinsic capacity to control their responsiveness to environmental signals that can be fluctuating and noisy. Using a novel single‐cell microRNA activity reporter, we show that miR‐142 is bimodally expressed in embryonic stem cells, creating two states indistinguishable by pluripotency markers.
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  • How to do a Proper Cell Culture Quick Check

    In order to successfully work with mammalian cell lines, they must be grown under controlled conditions and require their own specific growth medium. In addition, to guarantee consistency their growth must be monitored at regular intervals. This article describes a typical workflow for subculturing an adherent cell line with detailed illustrations of all of the necessary steps.
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  • Cross-strand Binding of TFAM to a Single mtDNA Molecule Forms the Mitochondrial Nucleoid

    Scientists from three Max Planck Institutes have gained fundamental insights into the organization of mitochondrial DNA (mtDNA). The researchers observed in high-resolution images gained with nobel prize-winning microscopy techniques that single copies of mtDNA are packaged by a specialized protein into slightly elongated structures of circa 100 nm in length.
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  • Webinar: Dissecting Protein Dynamics in Living Cells by FRAP

    This webinar presented by Dr Marco Fritzsche, University of Oxford, and Jennifer Horner, PhD, Leica Microsystems, you will learn about how to use Fluorescence Recovery After Photo-bleaching (FRAP) microscopy to study protein dynamics.
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  • Video Talk by Roger Tsien: Fluorescent Proteins

    Live cell imaging has been revolutionized by the discovery of the green fluorescent protein (GFP). This lecture covers the history of GFP, how it folds and becomes fluorescent, how it has been mutated to produce additional colors (blue, cyan, yellow), and the discovery of red fluorescent proteins from corals. It also covers novel photoswitchable and photoactivatible fluorescent proteins, whose color can be changed by light, and new infrared fluorescent proteins.
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  • Fluorescent Proteins Illuminate Cell Biology

    Green fluorescent protein (GFP) isolated from the jellyfish Aequorea victoria and GFP-like fluorescent proteins from other animals have had an important role in the technical innovations that have driven these advances. This poster provides a comprehensive user's guide to fluorescent proteins and sensors , their key properties and the cell biological questions to which they can be applied.
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  • "We can go home and the imaging is done automatically by the Leica HCS A Matrix Screener."

    Jutta Maria Bulkescher is the technical coordinator in the Novo Nordisk Foundation Center for Protein Research and Danish Stem Cell Center in Copenhagen, Denmark. The Leica HCS-A matrix screener is an invaluable tool for her facility. "It just gives us the biggest and easiest flexibility we can have to set up different imaging paramters and to check different conditions on one multi-well plate", explains Bulkescher.
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  • Video Talk by Joseph Gall: Early History of Microscopy

    Joseph Gall takes us through the history of early microscopes and the discovery of the cell. Compound microscopes were invented alongside the telescope in the 17th century; however these microscopes were not widely used until the late 19th century due to optical aberrations. In the meantime, simple microscopes were used throughout the 1700s and 1800s to make major discoveries in biology, including the first descriptions of the nucleus, cilia, cells, bacteria, and protozoans. Once optics improved in the mid to late 1800s, compound microscopes were used to discover chromosomes, mitosis, and other cellular structures.
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  • Webinar: Applications and Methodology of Super-Resolution Microscopy with Leica SR GSD 3D

    This webinar, sponsored by Leica Microsystems, will highlight applications in super-resolution microscopy. Researchers from the University of Washington and the federal University of São Paulo will share their experiences using the Ground State Depletion (GSD) method of super-resolution imaging to address diverse research topics such as the role of calcium signaling and excitation in cardiac muscle, and the structure and functional relationship of biological macromolecules.
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  • Cortical Actin Networks Induce Spatio-temporal Confinement of Phospholipids in the Plasma Membrane – A Minimally Invasive Investigation by STED-FCS

    Important discoveries in the last decades have changed our view of the plasma membrane organisation. Specifically, the cortical cytoskeleton has emerged as a key modulator of the lateral diffusion of membrane proteins. Cytoskeleton-dependent compartmentalised lipid diffusion has been proposed, but this concept remains controversial because this phenomenon has thus far only been observed with artefact-prone probes in combination with a single technique: single particle tracking.
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  • Abstracts of the 5th European Super-Resolution User-Club Meeting

    The 5th Super-Resolution User Club Meeting was held in collaboration with Professor Kees Jalink and The Netherlands Cancer Institute (NKI) in Amsterdam. Having the meeting at a location where super-resolution microscopy is used on a daily basis makes a big difference, offering participants the chance to use live cells for workshops and see systems working in their true environments. Thanks also to the scientists that supported the meeting by coming and giving talks. As super-resolution continues to grow in importance in research, we recognize the need to come together to network, share information and experiences. Here we present the abstracts of the talks.
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  • Webinar: Unleashing the Powers of Super-Resolution Microscopy to Solve Immunological Challenges

    In this webinar, Christian Eggeling and Dongfang Liu will discuss their experiences using STED super-resolution microscopy to explore, uncover and define the intricate machinery involved in immunological pathways and infectious disorders.
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  • Correlating Intravital Multi-Photon Microscopy to 3D Electron Microscopy of Invading Tumor Cells Using Anatomical Reference Points

    Cancer research unsing multiphoton microscopy and 3D electron microscopy. Correlative microscopy combines the advantages of both light and electron microscopy to enable imaging of rare and transient events at high resolution. Performing correlative microscopy in complex and bulky samples such as an entire living organism is a time-consuming and error-prone task.
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  • Immersion Freezing for Cryo-Transmission Electron Microscopy: Applications

    A well established usage case for cryo-TEM is three-dimensional reconstruction of isolated macromolecules, virus particles, or filaments. On one hand, these approaches are based on averaging of repetitive structures – either due to numerous identical molecules, repetitive patterns on a filament, or symmetries, to reduce the noise inherent to cryo-TEM.
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