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ASTM F2998-14

(Guide)

Guide for Using Fluorescence Microscopy to Quantify the Spread Area of Fixed Cells (Withdrawn 2023)

Guide for Using Fluorescence Microscopy to Quantify the Spread Area of Fixed Cells (Withdrawn 2023)

SIGNIFICANCE AND USE
5.1 Under well-controlled conditions, the quantitative evaluation of morphological features of a cell population can be used to identify changes in cellular behavior or state. Cell morphology changes may be expected when, for example, there is a response to changes in cellular cytoskeleton organization (1), a response of cells to toxic compounds, changes in differentiation state, and changes in adhesion properties of cells to a substrate by either chemical or mechanical-induced extracellular matrix-based (ECM-based) signaling pathways (2, 3). Typically, populations of cells exhibit a range of morphologies even when the cells are genetically identical and are in a homogeneous environment (4). This biological variation in cell response is due to both cell-cycle variations and stochasticity in the cellular reactions that control adhesion and spreading in cells. By using cell-by-cell, microscopy-based measurements and appropriate statistical sampling procedures, the distribution of cell morphologies such as cell spreading area per cell can be measured. This distribution is highly characteristic of the culture and conditions being examined.  
5.2 It is important to note that the use of this technique for cells on or in a 3-D scaffold materials can complicate the interpretation of the data. The topographic transforms of the cells on a 3-D material may require full volumetric imaging and not just wide-field fluorescence imaging as described here.  
5.3 the following are several examples of how this measurement can be used in a laboratory:—  
5.3.1 Quantify Cellular Response to a Biomaterial—The measurement of cell spread area can be used to characterize the response of cells to biomaterials. For example, spreading of most cell types is extremely sensitive to the stiffness of the culture substrate (5), (6). It is important to note that cell response to an ECM may be dependent on the preparation of the matrix. For example, the same ECM proteins prepared in a fibrillar or...
SCOPE
1.1 This 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.  
1.2 The 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.  
1.3 This 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.  
1.4 Cell 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 ...

General Information

Status
Withdrawn
Publication Date
31-Dec-2013
Withdrawal Date
05-Jan-2023
ICS
07.080 - Biology. Botany. Zoology
07.100.01 - Microbiology in general
Technical Committee
F04 - Medical and Surgical Materials and Devices
Drafting Committee
F04.46 - Cell Signaling
Current Stage
Ref Project

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ASTM F2998-14 - Guide for Using Fluorescence Microscopy to Quantify the Spread Area of Fixed Cells (Withdrawn 2023)ASTM F2998-14 - Guide for Using Fluorescence Microscopy to Quantify the Spread Area of Fixed Cells (Withdrawn 2023)
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ASTM F2998-14 - Guide for Using Fluorescence Microscopy to Quantify the Spread Area of Fixed Cells (Withdrawn 2023)
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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: F2998 − 14
Standard Guide for
Using Fluorescence Microscopy to Quantify the Spread
1
Area of Fixed Cells
This standard is issued under the fixed designation F2998; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope 1.5 This standard does not purport to address all of the
safety concerns, if any, associated with its use. It is the
1.1 This guide describes several measurement and technical
responsibility of the user of this standard to establish appro-
issues involved in quantifying the spread area of fixed cells.
priate safety and health practices and determine the applica-
Cell spreading and the distribution of cell spread areas of a
bility of regulatory limitations prior to use.
population of cells are the result of a biological response that
1.5.1 Sodium azide is used as a anti-bacterial reagent in the
is dependent on intracellular signaling mechanisms and the
slide mounting media. This preserves the integrity of the
characteristics of cell adhesion to a surface. Cell spread area is
mounting media. The toxicity of this reagent (for example,
a morphological feature that can be responsive to alteration in
MSDS) should be considered before use of this reagent in large
themetabolicstateorthestateofstressofthecells.Changesin
scale slide mounting procedures.
cell spread area can also indicate an alteration in the adhesion
substratethatmaybeduetodifferencesinmanufacturingofthe
2. Referenced Documents
substrate material or be in response to extracellular matrix
2.1 ASTM Standards:
secretions. High quality measurement of cell spread area can
E1488GuideforStatisticalProcedurestoUseinDeveloping
serve as a useful metric for benchmarking and detecting
and Applying Test Methods
changes cell behavior under experimental conditions.
F2150Guide for Characterization and Testing of Biomate-
1.2 The measurement described in this document is based
rial Scaffolds Used in Regenerative Medicine and Tissue-
on the use of fluorescence microscopy imaging of fixed cells
Engineered Medical Products
and the use of image analysis algorithms to extract relevant
data from the images. To produce robust cell spread area 3. Terminology
measurements, technical details involved in sample
3.1 Definition of Terms:
preparation, cell staining, microscopy imaging, image analysis
3.1.1 cell morphology—the physical shape properties of a
and statistical analysis should be considered. Several of these
cell such as cell volume, cell spread area, and cell perimeter;
issues are discussed within this document.
and the non-unit measures of roundness and circularity.
1.3 Thisstandardismeanttoserveasaguidefordeveloping
3.1.2 cell spread area—the area that encompasses a
methods to reliably measure the area to which cells spread at a
2-dimensional (2-D) projection of a 3-dimensional (3-D)
surface. This surface can be conventional tissue culture poly-
adhered cell.
styrene or sophisticated engineered biomaterial surfaces. An
3.1.3 cell object—a single cell or two or more adhered cells
example of a detailed procedure to measure the spreading area
adjacent to each other such that they cannot unambiguously be
of cells on a tissue culture polystyrene surface is provided in
segmentedfromoneanotherbycelledgedetectiontechniques.
the appendix section.
3.2 Definitions of Terms Specific to This Standard:
1.4 Cell morphology features such as cell spreading area
3.2.1 segmentation—the act of classifying pixels in an
and perimeter are generally reported in units of length. For
imageascellornon-cellareasandthegroupingofadjacentcell
example, spreading area per cell (that is, cell spread area) is
pixels into a cell object.
2
likely reported in units of µm .Aspatial calibration standard is
required to convert between numbers of pixels in a CCD
4. Summary of Practice
2
camera image to µm as an SI unit.
4.1 The measurement of a cell morphology feature such as
cell spread area in a population of cells in culture can be a
1
This guide is under the jurisdiction ofASTM Committee F04 on Medical and
quantitative characteristic of cell population behavior and cell
Surgical Materials and Devices and is the direct responsibility of Subcommittee
population state.This document provides guidance on measur-
F04.46 on Cell Signaling.
ing the 2-D morphological property (that is, cell spread area)
Current edition approved Jan. 1, 2014. Published May 2014. DOI: 10.1520/
F2998-14. for a population of cells in culture on a material.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
1

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4.1 The primary use of these test methods is testing to determine the specified mechanical properties of steel, stainless steel, and related alloy products for the evaluation of conformance of such products to a material specification under the jurisdiction of ASTM Committee A01 and its subcommittees as designated by a purchaser in a purchase order or contract.  
4.1.1 These test methods may be and are used by other ASTM Committees and other standards writing bodies for the purpose of conformance testing.  
4.1.2 The material condition at the time of testing, sampling frequency, specimen location and orientation, reporting requirements, and other test parameters are contained in the pertinent material specification or in a general requirement specification for the particular product form.  
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4.2 These test methods are also suitable to be used for testing of steel, stainless steel and related alloy materials for other purposes, such as incoming material acceptance testing by the purchaser or evaluation of components after service exposure.  
4.2.1 As with any mechanical testing, deviations from either specification limits or expected as-manufactured properties can occur for valid reasons besides deficiency of the original as-fabricated product. These reasons include, but are not limited to: subsequent service degradation from environmental exposure (for example, temperature, corrosion); static or cyclic service stress effects, mechanically-induced damage, material inhomogeneity, anisotropic structure, natural aging of select alloys, further processing not included in the specification, sampling limitations, and measuring equipment calibration uncertainty. There is statistical variation in all aspects of mechanical testin...
SCOPE
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Sections  
Tension  
7 to 14  
Bend  
15  
Hardness  
16  
Brinell  
17  
Rockwell  
18  
Portable  
19  
Impact  
20 to 30  
Keywords  
32  
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Annex  
Bar Products  
Annex A1  
Tubular Products  
Annex A2  
Fasteners  
Annex A3  
Round Wire Products  
Annex A4  
Significance of Notched-Bar Impact Testing  
Annex A5  
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Annex A6    
Testing Multi-Wire Strand  
Annex A7  
Rounding of Test Data  
Annex A8  
Methods for Testing Steel Reinforcing Bars  
Annex A9  
Procedure for Use and Control of Heat-cycle Simulation  
Annex A10  
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SCOPE
1.1 This specification covers cold expansion fittings and cross-linked (PEX) reinforcing rings for use with cross-linked polyethylene (PEX) plastic tubing in nominal tubing sizes of 3/8 , 1/2 , 5/8 , 3/4 , 1, 11/4 , 11/2 , 2, 21/2 , and 3 in. that meet the requirements of Specification F876 or F3253 and for use with Polyethylene of Raised Temperature (PE-RT) pipe in nominal tubing sizes of 3/8 , 1/2 , 5/8 , 3/4 , 1, 11/4 , 11/2 , 2, 21/2 , and 3 that meet the requirements of Specification F2769. These fittings are intended for use in 100 psi (690 kPa) cold- and hot-water distribution systems operating at temperatures up to and including 180 °F (82 °C). The system is comprised of a PEX reinforcing ring and a cold expansion fitting. Included are the requirements for materials, workmanship, dimensions, and markings to be used on the fitting components. The components covered by this specification are intended for use in residential and commercial, hot and cold, potable water distribution systems as well as building supply lines, sealed central heating, including under-floor-heating systems.  
1.2 The values stated in inch-pound units are to be regarded as standard. The values given in parentheses are mathematical conversions to SI units that are provided for information only and are not considered standard.  
1.3 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, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.4 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

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ASTM
ASTM E2521-23(Terminology)
Standard Terminology for Evaluating Response Robot Capabilities

SCOPE
1.1 This terminology identifies and precisely defines terms as used in the standard test methods, practices, and guides for evaluating response robots intended for hazardous environments. Further discussions of the terms can be found within the standards in which the terms appear.  
1.2 The term definitions address response robots, including ground, aquatic, and aerial systems. Some key features of such systems are remotely operated from safe standoff distances, deployable at operational tempos, capable of operating in complex environments, sufficiently hardened against harsh environments, reliable and field serviceable, durable or cost effectively disposable, and equipped with operational safeguards.  
1.3 Units—Values stated in either the International System of Units (metric) or U.S. Customary units (inch-pound) are to be regarded separately as standard. The values stated in each system may not be exact equivalents. Both units are referenced to facilitate acquisition of materials internationally and minimize fabrication costs. Tests conducted using either system maintain repeatability and reproducibility of the test method and results are comparable.  
1.4 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

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ASTM
ASTM C1306/C1306M-08(2023)(Test Method)
Standard Test Method for Hydrostatic Pressure Resistance of a Liquid-Applied Waterproofing Membrane

SIGNIFICANCE AND USE
5.1 This test method is used as a screening tool to determine the hydrostatic pressure to which a liquid-applied waterproofing membrane may be subjected without failing when stretched over a crack in the substrate. This test method discriminates between a membrane that is very resistant to hydrostatic pressure and one that is not. Because of the variability inherent in this test method, it is not recommended that this test method be used to set a numerical standard for hydrostatic pressure resistance. No prediction of durability at lower hydrostatic pressures can be made when using the results of this test method.
SCOPE
1.1 This test method describes a laboratory procedure for determining the resistance of a waterproofing membrane to hydrostatic pressure.  
1.2 The committee with jurisdiction over this standard is not aware of any comparable standards published by other organizations.  
1.3 There are no ISO standards similar or equivalent to this ASTM standard.  
1.4 The values stated in either SI units or inch-pound units are to be regarded separately as standard. The values stated in each system may not be exact equivalents; therefore, each system shall be used independently of the other. Combining values from the two systems may result in nonconformance with the standard.  
1.5 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, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.6 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

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