ASTM_E_1881_-_12.pdf

Designation:E188112Standard Guide forCell Culture Analysis with SIMS1This standard is issued under the fixed designation E1881;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 provides the Secondary Ion Mass Spectrom-etry(SIMS)analyst with a cryogenic method for analyzingindividual tissue culture cells growing in vitro.This guide issuitable for frozen-hydrated and frozen-freeze-dried sampletypes.Included are procedures for correlating optical,laserscanning confocal and secondary electron microscopies tocomplement SIMS analysis.1.2 This guide is not suitable for cell cultures that do notattach to the substrate.1.3 This guide is not suitable for any plastic embedded cellculture specimens.1.4 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.2.Referenced Documents2.1 ASTM Standards:2E673 Terminology Relating to Surface Analysis(Withdrawn2012)33.Terminology3.1 Definitions:3.1.1 See Terminology E673 for definitions of terms used inSIMS.4.Summary of Guide4.1 This guide describes a cryogenic freeze-fracture methodof sample preparation for cell culture specimens for SIMSanalysis.In brief,cell cultures are grown on a conductingsubstrate,such as silicon.When cells reach about 80%confluency,they are fast frozen and fractured by using asandwich method(1).4This allows freeze-fixation of cellularcontents and removal of the EF-leaflet of the apical plasmamembrane.Since this kind of fracture occurs in groups of cellsgrowing together,fractured cells are easily recognized foroptical,SEM and SIMS imaging.4.2 By correlative laser scanning confocal microscopy andSIMS,the same frozen freeze-dried cell can be analyzed fororganelle localization in relation to elemental content(2).5.Significance and Use5.1 The presence of cell growth medium complicates adirect analysis of cells with SIMS.Attempts to wash out thenutrient medium results in the exposure of cells to unphysi-ological reagents that may also alter their chemical composi-tion.This obstacle is overcome by using a sandwich freeze-fracture method(1).This cryogenic method has provided aunique way of sampling individual cells in their native state forSIMS analysis.5.2 The procedure described here has been successfullyused for imaging Na+and K+ion transport(3),calciumalterations in stimulated cells(4,5),and localization of thera-peutic drugs and isotopically labeled molecules in single cells(6).The frozen freeze-dried cells prepared according to thismethod have been checked for SIMS matrix effects(7).Ionimage quantification has also been achieved in this sample type(8).5.3 The procedure described here is amenable to a widevariety of cell cultures and provides a way for studying theresponse of individual cells for chemical alterations in the stateof health and disease and localization of isotopically-labeledmolecules and theraputic drugs in cell culture models.6.Apparatus6.1 This guide can be used for the analysis of cell cultureswith virtually any SIMS instrument.6.2 A cold stage in the SIMS instrument is needed toanalyze frozen-hydrated specimens(9).1This guide is under the jurisdiction of ASTM Committee E42 on SurfaceAnalysis and is the direct responsibility of Subcommittee E42.06 on SIMS.Current edition approved Nov.1,2012.Published December 2012.Originallyapproved in 1997.Last previous edition approved in 2006 as E1881 06.DOI:10.1520/E1881-12.2For referenced ASTM standards,visit the ASTM website,www.astm.org,orcontact ASTM Customer Service at serviceastm.org.For Annual Book of ASTMStandards volume information,refer to the standards Document Summary page onthe ASTM website.3The last approved version of this historical standard is referenced onwww.astm.org.4The boldface numbers in parentheses refer to a list of references at the end ofthis guide.Copyright ASTM International,100 Barr Harbor Drive,PO Box C700,West Conshohocken,PA 19428-2959.United States1 7.Procedure7.1 Cells are grown on silicon wafer pieces(approximately1 cm2area)of any shape.Alternatively,high purity germaniumwafer pieces are used for cell growth for studies involving theuse of44Ca stable isotope.These substrates are nontoxic tocells and have been used for growing various cell lines(1,2,8).Sterilize the silicon or germanium pieces prior to cell seeding.After the cells reach about 80%confluency,replace thenutrient growth medium with new medium containing 11 mpolystyrene beads(approximately 50 000 beads per 100 mmplastic dish,see Ref(1)for details on size of the beads).Thesebeads act as spacers during the sandwich-fracture tec