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  • ASTM F 2998 : 2014

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    Guide for Using Fluorescence Microscopy to Quantify the Spread Area of Fixed Cells (Withdrawn 2023)

    Available format(s):  Hardcopy, PDF

    Language(s):  English

    Published date:  17-05-2014

    Publisher:  American Society for Testing and Materials

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    Abstract - (Show below) - (Hide below)

    CONTAINED IN VOL. 13.02, 2015 Defines several measurement and technical issues involved in quantifying the spread area of fixed cells.

    Scope - (Show below) - (Hide below)

    1.1This guide describes several measurement and technical issues involved in quantifying the spread area of fixed cells. Cell spreading and the distribution of cell spread areas of a population of cells are the result of a biological response that is dependent on intracellular signaling mechanisms and the characteristics of cell adhesion to a surface. Cell spread area is a morphological feature that can be responsive to alteration in the metabolic state or the state of stress of the cells. Changes in cell spread area can also indicate an alteration in the adhesion substrate that may be due to differences in manufacturing of the substrate material or be in response to extracellular matrix secretions. High quality measurement of cell spread area can serve as a useful metric for benchmarking and detecting changes cell behavior under experimental conditions.

    1.2The measurement described in this document is based on the use of fluorescence microscopy imaging of fixed cells and the use of image analysis algorithms to extract relevant data from the images. To produce robust cell spread area measurements, technical details involved in sample preparation, cell staining, microscopy imaging, image analysis and statistical analysis should be considered. Several of these issues are discussed within this document.

    1.3This standard is meant to serve as a guide for developing methods to reliably measure the area to which cells spread at a surface. This surface can be conventional tissue culture polystyrene or sophisticated engineered biomaterial surfaces. An example of a detailed procedure to measure the spreading area of cells on a tissue culture polystyrene surface is provided in the appendix section.

    1.4Cell morphology features such as cell spreading area and perimeter are generally reported in units of length. For example, spreading area per cell (that is, cell spread area) is likely reported in units of µm2. A spatial calibration standard is required to convert between numbers of pixels in a CCD camera image to µm2 as an SI unit.

    1.5This 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.

    1.5.1Sodium azide is used as a anti-bacterial reagent in the slide mounting media. This preserves the integrity of the mounting media. The toxicity of this reagent (for example, MSDS) should be considered before use of this reagent in large scale slide mounting procedures.

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    Committee F 04
    Document Type Guide
    Publisher American Society for Testing and Materials
    Status Current

    Standards Referenced By This Book - (Show below) - (Hide below)

    ASTM F 3294 : 2018 Standard Guide for Performing Quantitative Fluorescence Intensity Measurements in Cell-based Assays with Widefield Epifluorescence Microscopy
    ASTM F 2944 : 2020 Standard Practice for Automated Colony Forming Unit (CFU) Assays—Image Acquisition and Analysis Method for Enumerating and Characterizing Cells and Colonies in Culture
    ASTM F 2739 : 2019 Standard Guide for Quantifying Cell Viability and Related Attributes within Biomaterial Scaffolds
    ASTM F 3088 : 2022 Standard Practice for Use of a Centrifugation Method to Quantify/Study Cell-Material Adhesive Interactions
    ASTM F 2739 : 2016 Standard Guide for Quantifying Cell Viability within Biomaterial Scaffolds

    Standards Referencing This Book - (Show below) - (Hide below)

    ASTM F 2150 : 2013 : REDLINE Standard Guide for Characterization and Testing of Biomaterial Scaffolds Used in Tissue-Engineered Medical Products
    ASTM E 1488 : 2009 Standard Guide for Statistical Procedures to Use in Developing and Applying Test Methods
    ASTM E 1488 : 2002 : EDT 1 Standard Guide for Statistical Procedures to Use in Developing and Applying Test Methods
    ASTM F 2150 : 2019 Standard Guide for Characterization and Testing of Biomaterial Scaffolds Used in Regenerative Medicine and Tissue-Engineered Medical Products
    ASTM E 1488 : 2008 Standard Guide for Statistical Procedures to Use in Developing and Applying Test Methods
    ASTM F 2150 : 2013 Standard Guide for Characterization and Testing of Biomaterial Scaffolds Used in Tissue-Engineered Medical Products
    ASTM F 2150 : 2002 : EDT 1 Standard Guide for Characterization and Testing of Biomaterial Scaffolds Used in Tissue-Engineered Medical Products
    ASTM E 1488 : 1996 Standard Guide for Statistical Procedures to Use in Developing and Applying ASTM Test Methods
    ASTM E 1488 : 2008 : REV A Standard Guide for Statistical Procedures to Use in Developing and Applying Test Methods
    ASTM E 1488 : 2012 : R2018 Standard Guide for Statistical Procedures to Use in Developing and Applying Test Methods
    ASTM E 1488 : 2012 : EDT 1 Standard Guide for Statistical Procedures to Use in Developing and Applying Test Methods
    ASTM F 2150 : 2007 Standard Guide for Characterization and Testing of Biomaterial Scaffolds Used in Tissue-Engineered Medical Products
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