{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,16]],"date-time":"2026-09-16T17:09:12Z","timestamp":1789578552559,"version":"build-2803163510"},"reference-count":30,"publisher":"Wiley","issue":"4","license":[{"start":{"date-parts":[[2024,4,11]],"date-time":"2024-04-11T00:00:00Z","timestamp":1712793600000},"content-version":"vor","delay-in-days":10,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Current Protocols"],"published-print":{"date-parts":[[2024,4]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>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 \u201cresident\u201d fibroblasts can undergo a transformation through a continuum of activation states into what is commonly known as a \u201cmyofibroblast,\u201d 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 <jats:italic>in vitro<\/jats:italic>. 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\u2010physiologic 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\u2010stiffness substrates, such as 8\u2010kPa polydimethylsiloxane (PDMS), has shown promise in reducing basal activation of fibroblasts. Here, we describe cell culture protocol for maintaining CF quiescence <jats:italic>in vitro<\/jats:italic> to enable a dynamic range for the assessment of activation status in response to fibrogenic stimuli using PDMS\u2010coated coverslips. Our protocol provides a cost\u2010effective tool to study fibroblast signaling and activity, allowing researchers to better understand the underlying mechanisms involved in cardiac fibrosis. \u00a9 2024 The Authors. Current Protocols published by Wiley Periodicals LLC.<\/jats:p><jats:p><jats:bold>Basic Protocol 1<\/jats:bold>: Generation of 8\u2010kPa polydimethylsiloxane (PDMS)\/gelatin\u2010coated coverslips for cardiac fibroblast cell culture<\/jats:p><jats:p><jats:bold>Basic Protocol 2<\/jats:bold>: Isolation of adult cardiac fibroblasts and plating onto PDMS coverslips<\/jats:p><jats:p><jats:bold>Basic Protocol 3<\/jats:bold>: Assessment of cardiac fibroblast activation by \u03b1 smooth muscle actin (\u03b1SMA) immunocytochemistry<\/jats:p>","DOI":"10.1002\/cpz1.1025","type":"journal-article","created":{"date-parts":[[2024,4,11]],"date-time":"2024-04-11T07:19:05Z","timestamp":1712819945000},"source":"Crossref","is-referenced-by-count":4,"title":["<i>In Vitro<\/i> Assessment of Cardiac Fibroblast Activation at Physiologic Stiffness"],"prefix":"10.1002","volume":"4","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-8292-6330","authenticated-orcid":false,"given":"Robert S.","family":"Goldsmith","sequence":"first","affiliation":[{"name":"Department of Pharmacology University of Michigan  Ann Arbor Michigan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4904-7670","authenticated-orcid":false,"given":"Yao\u2010Chang","family":"Tsan","sequence":"additional","affiliation":[{"name":"Department of Internal Medicine University of Michigan  Ann Arbor Michigan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rachel E.","family":"Scissors","sequence":"additional","affiliation":[{"name":"Department of Pharmacology University of Michigan  Ann Arbor Michigan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Adam S.","family":"Helms","sequence":"additional","affiliation":[{"name":"Department of Internal Medicine University of Michigan  Ann Arbor Michigan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Matthew 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