ASTM E1881-97(2002)

Standard Guide for Cell Culture Analysis with SIMS

SCOPE
1.1 This guide provides the Secondary Ion Mass Spectrometry (SIMS) analyst with a cryogenic method for analyzing individual tissue culture cells growing in vitro. This guide is suitable for frozen-hydrated and frozen-freeze-dried sample types. Included are procedures for correlating optical, laser scanning confocal and secondary electron microscopies to compliment SIMS analysis.
1.2 This guide is not suitable for cell cultures that do not attach to the substrate.
1.3 This guide is not suitable for any plastic embedded cell culture specimens.
1.4 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.

General Information

• Status: Historical
• Publication Date: 09-May-1997
• ICS: 07.100.10 - Medical microbiology
• Technical Committee: E42 - Surface Analysis
• Drafting Committee: E42.06 - SIMS

Relations

• Replaces: ASTM E1881-97 - Standard Guide for Cell Culture Analysis with SIMS
• Effective Date: 10-May-1997
• Replaced By: ASTM E1881-06 - Standard Guide for Cell Culture Analysis with SIMS
• Effective Date: 01-Nov-2006

Standards Content (Sample)

NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
Contact ASTM International (www.astm.org) for the latest information
Designation:E1881–97 (Reapproved 2002)
Standard Guide for
1
Cell Culture Analysis with SIMS
This standard is issued under the fixed designation E 1881; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (e) indicates an editorial change since the last revision or reapproval.
1. Scope 4.2 By correlative laser scanning confocal microscopy and
SIMS, the same frozen freeze-dried cell can be analyzed for
1.1 This guide provides the Secondary Ion Mass Spectrom-
organelle localization in relation to elemental content (2).
etry (SIMS) analyst with a cryogenic method for analyzing
individual tissue culture cells growing in vitro. This guide is
5. Significance and Use
suitable for frozen-hydrated and frozen-freeze-dried sample
5.1 The presence of cell growth medium complicates a
types. Included are procedures for correlating optical, laser
direct analysis of cells with SIMS. Attempts to wash out the
scanning confocal and secondary electron microscopies to
nutrient medium results in the exposure of cells to unphysi-
compliment SIMS analysis.
ological reagents that may also alter their chemical composi-
1.2 This guide is not suitable for cell cultures that do not
tion. This obstacle is overcome by using a sandwich freeze-
attach to the substrate.
fracture method (1). This cryogenic method has provided a
1.3 This guide is not suitable for any plastic embedded cell
uniquewayofsamplingindividualcellsintheirnativestatefor
culture specimens.
SIMS analysis.
1.4 This standard does not purport to address all of the
5.2 The procedure described here has been successfully
safety concerns, if any, associated with its use. It is the
+ +
used for imaging Na and K ion transport (3), calcium
responsibility of the user of this standard to establish appro-
alterations in stimulated cells (4,5), and localization of thera-
priate safety and health practices and determine the applica-
peutic drugs and isotopically labeled molecules in single cells
bility of regulatory limitations prior to use.
(6). The frozen freeze-dried cells prepared according to this
2. Referenced Documents method have been checked for SIMS matrix effects (7). Ion
imagequantificationhasalsobeenachievedinthissampletype
2.1 ASTM Standards:
2 (8).
E 673 Terminology Related to Surface Analysis
5.3 The procedure described here is amenable to a wide
3. Terminology variety of cell cultures and provides a way for studying the
response of individual cells for chemical alterations in the state
3.1 Definitions:
of health and disease.
3.1.1 SeeTerminologyE 673fordefinitionsoftermsusedin
SIMS.
6. Apparatus
4. Summary of Guide 6.1 This guide can be used for the analysis of cell cultures
with virtually any SIMS instrument.
4.1 This guide describes a cryogenic method of sample
6.2 A cold stage in the SIMS instrument is needed to
preparation for cell culture specimens for SIMS analysis. In
analyze frozen-hydrated specimens (9).
brief,cellculturesaregrownonaconductingsubstrate,suchas
silicon. When cells reach about 80 % confluency, they are fast
7. Procedure
3
frozen and fractured by using a sandwich method (1). This
7.1 Cells are grown on silicon wafer pieces (approximately
allows freeze-fixation of cellular contents and removal of the
2
1cm area) of any shape.Alternatively, high purity germanium
EF-leaflet of the apical plasma membrane. Since this kind of
wafer pieces are used for cell growth for studies involving the
fracture occurs in groups of cells growing together, fractured
44
useof Castableisotope.Thesesubstratesarenontoxictocells
cells are easily recognized for optical, SEM and SIMS imag-
and have been used for growing various cell lines (1,2,8).
ing.
Sterilize the silicon or germanium pieces prior to cell seeding.
After the cells reach about 80 % confluency, replace the
1
This guide is under the jurisdiction of ASTM Committee E42 on Surface
nutrient growth medium with new medium containing 11 µm
Analysis and is the direct responsibility of Subcommittee E 42.06 on SIMS.
polystyrene beads (approximately 50 000 beads per 100 mm
Current edition approved May 10, 1997. Published July 1997.
2 plastic dish, see Ref (1) for details on size of the beads). These
Annual Book of ASTM Standards, Vol 03.06.
3
beads act as spacers during the sandwich-fracture technique. It
The boldface numbers in parentheses refer to a list of references at the end of
this guide.
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E1881–97 (2002)
takes approximately 30 min for the beads to settle down on the thesiliconsubstratearenowreadyforfrozen-hydratedana
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