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Description of ASTM-F2997 2013ASTM F2997-13Historical Standard: Standard Practice for Quantification of Calcium Deposits in Osteogenic Culture of Progenitor Cells Using Fluorescent Image AnalysisASTM F2997Scope 1.1 This practice defines a method for the estimation of calcium content at multiple time points in living cell cultures that have been cultured under conditions known to promote mineralization. The practice involves applying a fluorescent calcium chelating dye that binds to the calcium phosphate mineral crystals present in the live cultures followed by image analysis of fluorescence microscopy images of the stained cell cultures. Quantification of the positively stained areas provides a relative measure of the calcium content in the cell culture plate. A precise correlation between the image analysis parameters and calcium content is beyond the scope of this practice. 1.2 Calcium deposition in a secreted matrix is one of several features that characterize bone formation (in vitro and in vivo), and is therefore a parameter that may indicate bone formation and osteoblast function (i.e., osteoblastic differentiation). Calcium deposition may, however, be unrelated to osteoblast differentiation status if extensive cell death occurs in the cell cultures or if high amounts of osteogenic medium components that lead to artifactual calcium-based precipitates are used. Distinguishing between calcium deposition associated with osteoblast-produced mineralized matrix and that from pathological or artifactual deposition requires additional structural and chemical characterization of the mineralized matrix and biological characterization of the cell that is beyond the scope of this practice. 1.3 The parameters obtained by image analysis are expressed in relative fluorescence units or area percentage, e.g., fraction of coverage of the area analyzed. 1.4 Units—The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard. 1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use. Keywords bone; calcification; image analysis; mineralization; osteoblast; stem cell; tissue engineering; xylenol orange ICS Code ICS Number Code 07.100.10 (Medical microbiology) DOI: 10.1520/F2997-13 The following editions for this book are also available...This book also exists in the following packages...Subscription InformationMADCAD.com ASTM Standards subscriptions are annual and access is unlimited concurrency based (number of people that can access the subscription at any given time) from single office location. For pricing on multiple office location ASTM Standards Subscriptions, please contact us at info@madcad.com or +1 800.798.9296.
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About ASTMASTM International, formerly known as the American Society for Testing and Materials (ASTM), is a globally recognized leader in the development and delivery of international voluntary consensus standards. Today, some 12,000 ASTM standards are used around the world to improve product quality, enhance safety, facilitate market access and trade, and build consumer confidence. ASTM’s leadership in international standards development is driven by the contributions of its members: more than 30,000 of the world’s top technical experts and business professionals representing 150 countries. Working in an open and transparent process and using ASTM’s advanced electronic infrastructure, ASTM members deliver the test methods, specifications, guides, and practices that support industries and governments worldwide. |
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