ASTM E1881-97

Standard Guide for Cell Culture Analysis with SIMS

SCOPE
1.1 This guide provides the Secondary Ion Mass Spectometry (SIMS) analyst with a cryogenic method for analyzing individual tissue culture cells growing into 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
• Technical Committee: E42 - Surface Analysis
• Drafting Committee: E42.06 - SIMS

Relations

• Replaced By: ASTM E1881-97(2002) - Standard Guide for Cell Culture Analysis with SIMS
• Effective Date: 10-May-1997

Standards Content (Sample)

NOTICE: This standard has either been superseded and replaced by a new version or discontinued.
Contact ASTM International (www.astm.org) for the latest information.
Designation: E 1881 – 97
Standard Guide for
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 SIMS, the same frozen freeze-dried cell can be analyzed for
organelle localization in relation to elemental content (2).
1.1 This guide provides the Secondary Ion Mass Spectrom-
etry (SIMS) analyst with a cryogenic method for analyzing
5. Significance and Use
individual tissue culture cells growing in vitro. This guide is
5.1 The presence of cell growth medium complicates a
suitable for frozen-hydrated and frozen-freeze-dried sample
direct analysis of cells with SIMS. Attempts to wash out the
types. Included are procedures for correlating optical, laser
nutrient medium results in the exposure of cells to unphysi-
scanning confocal and secondary electron microscopies to
ological reagents that may also alter their chemical composi-
compliment SIMS analysis.
tion. This obstacle is overcome by using a sandwich freeze-
1.2 This guide is not suitable for cell cultures that do not
fracture method (1). This cryogenic method has provided a
attach to the substrate.
unique way of sampling individual cells in their native state for
1.3 This guide is not suitable for any plastic embedded cell
SIMS analysis.
culture specimens.
5.2 The procedure described here has been successfully
1.4 This standard does not purport to address all of the
+ +
used for imaging Na and K ion transport (3), calcium
safety concerns, if any, associated with its use. It is the
alterations in stimulated cells (4,5), and localization of thera-
responsibility of the user of this standard to establish appro-
peutic drugs and isotopically labeled molecules in single cells
priate safety and health practices and determine the applica-
(6). The frozen freeze-dried cells prepared according to this
bility of regulatory limitations prior to use.
method have been checked for SIMS matrix effects (7). Ion
2. Referenced Documents image quantification has also been achieved in this sample type
(8).
2.1 ASTM Standards:
5.3 The procedure described here is amenable to a wide
E 673 Terminology Related to Surface Analysis
variety of cell cultures and provides a way for studying the
3. Terminology
response of individual cells for chemical alterations in the state
of health and disease.
3.1 Definitions:
3.1.1 See Terminology E 673 for definitions of terms used in
6. Apparatus
SIMS.
6.1 This guide can be used for the analysis of cell cultures
4. Summary of Guide
with virtually any SIMS instrument.
6.2 A cold stage in the SIMS instrument is needed to
4.1 This guide describes a cryogenic method of sample
analyze frozen-hydrated specimens (9).
preparation for cell culture specimens for SIMS analysis. In
brief, cell cultures are grown on a conducting substrate, such as
7. Procedure
silicon. When cells reach about 80 % confluency, they are fast
7.1 Cells are grown on silicon wafer pieces (approximately
frozen and fractured by using a sandwich method (1). This
1cm area) of any shape. Alternatively, high purity germanium
allows freeze-fixation of cellular contents and removal of the
wafer pieces are used for cell growth for studies involving the
EF-leaflet of the apical plasma membrane. Since this kind of
use of Ca stable isotope. These substrates are nontoxic to cells
fracture occurs in groups of cells growing together, fractured
and have been used for growing various cell lines (1,2,8).
cells are easily recognized for optical, SEM and SIMS imag-
Sterilize the silicon or germanium pieces prior to cell seeding.
ing.
After the cells reach about 80 % confluency, replace the
4.2 By correlative laser scanning confocal microscopy and
nutrient growth medium with new medium containing 11 μm
polystyrene beads (approximately 50 000 beads per 100 mm
This guide is under the jurisdiction of ASTM Committee E-42 on Surface
plastic dish, see Ref (1) for details on size of the beads). These
Analysis and is the direct responsibility of Subcommittee E 42.06 on SIMS.
beads act as spacers during the sandwich-fracture technique. It
Current edition approved May 10, 1997. Published July 1997.
2 takes approximately 30 min for the beads to settle down on the
Annual Book of ASTM Standards, Vol 03.06.
substrate. After beads settle down on the substrate the cells can
The boldface numbers in parentheses refer to a list of references at the end of
this guide.
Copyright © ASTM, 100 Barr Harbor Drive, West Conshohocken, PA 19428-2959, United States.
NOTICE: This standard has either been superseded and replaced by a new version or discontinued.
Contact ASTM International (www.astm.org) for the latest information.
E 1881
be subjected to desired treatments and cryogenic sampling. 7.1.2 Depending on the need of a particular SIMS analysis,
7.1.1 After the desired treatments fast freeze and freeze- the freeze-dried cells may be analyze
...