ASTM_F_2998_-_14.pdf

Designation:F299814Guide forUsing Fluorescence Microscopy to Quantify the SpreadArea of Fixed Cells1This standard is issued under the fixed designation F2998;the number immediately following the designation indicates the year oforiginal adoption or,in the case of revision,the year of last revision.A number in parentheses indicates the year of last reapproval.Asuperscript epsilon()indicates an editorial change since the last revision or reapproval.1.Scope1.1 This guide describes several measurement and technicalissues involved in quantifying the spread area of fixed cells.Cell spreading and the distribution of cell spread areas of apopulation of cells are the result of a biological response thatis dependent on intracellular signaling mechanisms and thecharacteristics of cell adhesion to a surface.Cell spread area isa morphological feature that can be responsive to alteration inthe metabolic state or the state of stress of the cells.Changes incell spread area can also indicate an alteration in the adhesionsubstrate that may be due to differences in manufacturing of thesubstrate material or be in response to extracellular matrixsecretions.High quality measurement of cell spread area canserve as a useful metric for benchmarking and detectingchanges cell behavior under experimental conditions.1.2 The measurement described in this document is basedon the use of fluorescence microscopy imaging of fixed cellsand the use of image analysis algorithms to extract relevantdata from the images.To produce robust cell spread areameasurements,technicaldetailsinvolvedinsamplepreparation,cell staining,microscopy imaging,image analysisand statistical analysis should be considered.Several of theseissues are discussed within this document.1.3 This standard is meant to serve as a guide for developingmethods to reliably measure the area to which cells spread at asurface.This surface can be conventional tissue culture poly-styrene or sophisticated engineered biomaterial surfaces.Anexample of a detailed procedure to measure the spreading areaof cells on a tissue culture polystyrene surface is provided inthe appendix section.1.4 Cell morphology features such as cell spreading areaand perimeter are generally reported in units of length.Forexample,spreading area per cell(that is,cell spread area)islikely reported in units of m2.A spatial calibration standard isrequired to convert between numbers of pixels in a CCDcamera image to m2as an SI unit.1.5 This standard does not purport to address all of thesafety concerns,if any,associated with its use.It is theresponsibility of the user of this standard to establish appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use.1.5.1 Sodium azide is used as a anti-bacterial reagent in theslide mounting media.This preserves the integrity of themounting media.The toxicity of this reagent(for example,MSDS)should be considered before use of this reagent in largescale slide mounting procedures.2.Referenced Documents2.1 ASTM Standards:E1488 Guide for Statistical Procedures to Use in Developingand Applying Test MethodsF2150 Guide for Characterization and Testing of Biomate-rial Scaffolds Used in Tissue-Engineered Medical Prod-ucts3.Terminology3.1 Definition of Terms:3.1.1 cell morphologythe physical shape properties of acell such as cell volume,cell spread area,and cell perimeter;and the non-unit measures of roundness and circularity.3.1.2 cell spread areathe area that encompasses a2-dimensional(2-D)projection of a 3-dimensional(3-D)adhered cell.3.1.3 cell objecta single cell or two or more adhered cellsadjacent to each other such that they cannot unambiguously besegmented from one another by cell edge detection techniques.3.2 Definitions of Terms Specific to This Standard:3.2.1 segmentationthe act of classifying pixels in animage as cell or non-cell areas and the grouping of adjacent cellpixels into a cell object.4.Summary of Practice4.1 The measurement of a cell morphology feature such ascell spread area in a population of cells in culture can be aquantitative characteristic of cell population behavior and cellpopulation state.This document provides guidance on measur-ing the 2-D morphological property(that is,cell spread area)for a population of cells in culture on a material.1This test method is under the jurisdiction ofASTM Committee F04 on Medicaland Surgical Materials and Devices and is the direct responsibility of SubcommitteeF04.46 on Cell Signaling.Current edition approved Jan.1,2014.Published May 2014.DOI:10.1520/F2998-14.Copyright ASTM International,100 Barr Harbor Drive,PO Box C700,West Conshohocken,PA 19428-2959.United States1 4.2 This measurement is typically achieved by seeding apopulation of cells at low density on a test surface andchemically stabilizing the cellular structure by fixing the cellsat a particular point in time.The cells are then treated with twostains:one that discriminates the cell from non-cellbackground,and anothe