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换一换In Vitro Assessment of Cardiac Fibroblast Activation at Physiologic Stiffness
Robert S. Goldsmith, Yao-Chang Tsan, Rachel E. Scissors, Adam S. Helms, Matthew J. Brody, Robert S. Goldsmith, Yao-Chang Tsan, Rachel E. Scissors, Adam S. Helms, Matthew J. Brody
Cardiac fibroblasts (CF) are an essential cell type in cardiac physiology, playing diverse roles in maintaining structural integrity, extracellular matrix (ECM) synthesis, and tissue repair. Under normal conditions, these cells reside in the interstitium in a quiescent state poised to sense and respond to injury by synthesizing and secreting collagen, vimentin, hyaluronan, and other ECM components. In response to mechanical and chemical stimuli, these “resident” fibroblasts can undergo a transformation through a continuum of activation states into what is commonly known as a “myofibroblast,” in a process critical for injury response. Despite progress in understanding the contribution of fibroblasts to cardiac health and disease, much remains unknown about the signaling mediating this activation, in part owing to technical challenges in evaluating CF function and activation status _in vitro_. Given their role in monitoring the ECM, CFs are acutely sensitive to stiffness and pressure. High basal activation of isolated CFs is common due to the super-physiologic stiffness of traditional cell culture substrates, making assays dependent on quiescent cells challenging. To overcome this problem, cell culture parameters must be tightly controlled, and the use of dishes coated with biocompatible reduced-stiffness substrates, such as 8-kPa polydimethylsiloxane (PDMS), has shown promise in reducing basal activation of fibroblasts. Here, we describe cell culture protocol for maintaining CF quiescence _in vitro_ to enable a dynamic range for the assessment of activation status in response to fibrogenic stimuli using PDMS-coated coverslips. Our protocol provides a cost-effective tool to study fibroblast signaling and activity, allowing researchers to better understand the underlying mechanisms involved in cardiac fibrosis. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. **Basic Protocol 1** : Generation of 8-kPa polydimethylsiloxane (PDMS)/gelatin-coated coverslips for cardiac fibroblast cell culture **Basic Protocol 2** : Isolation of adult cardiac fibroblasts and plating onto PDMS coverslips **Basic Protocol 3** : Assessment of cardiac fibroblast activation by α smooth muscle actin (αSMA) immunocytochemistry
AI 解读Packaging and containerization of computational methods
Mohammed Alser, Brendan Lawlor, Richard J. Abdill, Sharon Waymost, Ram Ayyala, Neha Rajkumar, Nathan LaPierre, Jaqueline Brito, André M. Ribeiro-dos-Santos, Nour Almadhoun, Varuni Sarwal, Can Firtina, Tomasz Osinski, Eleazar Eskin, Qiyang Hu, Derek Strong, Byoung-Do (B.D) Kim, Malak S. Abedalthagafi, Onur Mutlu, Serghei Mangul
Methods for analyzing the full complement of a biomolecule type, e.g., proteomics or metabolomics, generate large amounts of complex data. The software tools used to analyze omics data have reshaped the landscape of modern biology and become an essential component of biomedical research. These tools are themselves quite complex and often require the installation of other supporting software, libraries and/or databases. A researcher may also be using multiple different tools that require different versions of the same supporting materials. The increasing dependence of biomedical scientists on these powerful tools creates a need for easier installation and greater usability. Packaging and containerization are different approaches to satisfy this need by delivering omics tools already wrapped in additional software that makes the tools easier to install and use. In this systematic review, we describe and compare the features of prominent packaging and containerization platforms. We outline the challenges, advantages and limitations of each approach and some of the most widely used platforms from the perspectives of users, software developers and system administrators. We also propose principles to make the distribution of omics software more sustainable and robust to increase the reproducibility of biomedical and life science research.
AI 解读Cell culture, transfection, immunocytochemistry, and imaging
Nisha Mohd Rafiq, Pietro De Camilli
This protocol describes the maintenance, transfection, immunocytochemistry, and imaging of RPE1 and also transfection, immunocytochemistry, and imaging of iPSCs, i<sup>3</sup>Neurons and DA neurons.
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