1、PD ISO/TS 19006:2016Nanotechnologies 5-(and 6)-Chloromethyl-2,7 Dichloro-dihydrofluorescein diacetate(CM-H2DCF-DA) assay forevaluating nanoparticle-induced intracellular reactiveoxygen species (ROS)production in RAW 264.7macrophage cell lineBSI Standards PublicationWB11885_BSI_StandardCovs_2013_AW.i
2、ndd 1 15/05/2013 15:06PD ISO/TS 19006:2016 PUBLISHED DOCUMENTNational forewordThis Published Document is the UK implementation of ISO/TS19006:2016.The UK participation in its preparation was entrusted to TechnicalCommittee NTI/1, Nanotechnologies.A list of organizations represented on this committee
3、 can beobtained on request to its secretary.This publication does not purport to include all the necessaryprovisions of a contract. Users are responsible for its correctapplication. The British Standards Institution 2016. Published by BSI StandardsLimited 2016ISBN 978 0 580 83224 6ICS 07.030Complian
4、ce with a British Standard cannot confer immunity fromlegal obligations.This Published Document was published under the authority of theStandards Policy and Strategy Committee on 31 August 2016.Amendments issued since publicationDate Text affectedPD ISO/TS 19006:2016 ISO 2016Nanotechnologies 5-(and
5、6)-Chloromethyl-2,7 Dichloro-dihydrofluorescein diacetate (CM-H2DCF-DA) assay for evaluating nanoparticle-induced intracellular reactive oxygen species (ROS) production in RAW 264.7 macrophage cell lineNanotechnologies Essai au diactate de 5-(et 6)- Chloromthyle -2,7 Dichloro-dihydro-fluorescine (CM
6、-H2DCF-DA) pour lvaluation de la gnration intracellulaire despces ractives loxygne induites par les nanoparticules sur la ligne souche 264.7 de macrophagesTECHNICAL SPECIFICATIONISO/TS19006Reference numberISO/TS 19006:2016(E)First edition2016-08-01PD ISO/TS 19006:2016ISO/TS 19006:2016(E)ii ISO 2016
7、All rights reservedCOPYRIGHT PROTECTED DOCUMENT ISO 2016, Published in SwitzerlandAll rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on the inte
8、rnet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below or ISOs member body in the country of the requester.ISO copyright officeCh. de Blandonnet 8 CP 401CH-1214 Vernier, Geneva, SwitzerlandTel. +41 22 749 01 11Fax +41 22 749 09 47copyr
9、ightiso.orgwww.iso.orgPD ISO/TS 19006:2016ISO/TS 19006:2016(E)Foreword ivIntroduction v1 Scope . 12 Normative references 13 Terms and definitions . 14 Symbols and abbreviated terms . 35 Materials . 36 Technical equipment 57 Nanoparticle sample preparation 58 Preparations . 68.1 General . 68.2 Flow c
10、ytometry calibration 78.3 Experimental culture medium 78.4 Reagent preparation. 78.5 Preparation of cell stock culture 78.6 Preparing culture for experiments 78.7 Verification of healthy cell growth . 88.8 Evaluation of nanoparticle interference . 98.9 Control preparation 98.9.1 General 98.9.2 Contr
11、ol description 98.9.3 Sin-1 stock solution preparation (1 mM) 109 Evaluation of nanoparticle impact on ROS generation in cells .109.1 Prepare cells in the 24 well plates . 109.2 Dose the cells with nanoparticles and controls .109.3 Expose the cells to CM-H2DCF-DA Assay . 119.4 Incubate the cells wit
12、h CM-H2DCF-DA 129.5 Flow cytometry analysis 1210 Data analysis and results 12Annex A (informative) Alternate cell lines 14Annex B (informative) Alternate fluorescence characterization techniques 15Annex C (informative) Suspension preparation and characterization 16Annex D (informative) Example exper
13、imental data from RAW 264.7 17Bibliography .20 ISO 2016 All rights reserved iiiContents PagePD ISO/TS 19006:2016ISO/TS 19006:2016(E)ForewordISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing Inter
14、national Standards is normally carried out through ISO technical committees. Each member body interested in a subject for which a technical committee has been established has the right to be represented on that committee. International organizations, governmental and non-governmental, in liaison wit
15、h ISO, also take part in the work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.The procedures used to develop this document and those intended for its further maintenance are described in the ISO/IEC Directives,
16、 Part 1. In particular the different approval criteria needed for the different types of ISO documents should be noted. This document was drafted in accordance with the editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).Attention is drawn to the possibility that some of t
17、he elements of this document may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of any patent rights identified during the development of the document will be in the Introduction and/or on the ISO list of patent declarations
18、received (see www.iso.org/patents).Any trade name used in this document is information given for the convenience of users and does not constitute an endorsement.For an explanation on the meaning of ISO specific terms and expressions related to conformity assessment, as well as information about ISOs
19、 adherence to the World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see the following URL: www.iso.org/iso/foreword.html.The committee responsible for this document is ISO/TC 229, Nanotechnologies.iv ISO 2016 All rights reservedPD ISO/TS 19006:2016ISO/TS 19006:2016(E
20、)IntroductionThe field of nanotechnology continues to advance rapidly through the development of new materials, products and applications. At the same time, many questions have been raised relating to the potential impact on human health and on the environment of some of these materials. Internation
21、ally, a large programme of research is underway to better understand and quantify potential hazards. Also, the chemicals used to coat the surface of nanoparticles in processing or in products can affect the interactions of nanoparticles with cells, even more so due to their large surface to volume r
22、atio. Thus, there is a need for reliable fast screening methods to determine the potential toxicity aspects of nanoparticles with characterization of chemical functionalization on nanoparticles.It is likely that monitoring biological response of cellular model systems to nanoparticle exposure can pr
23、ovide insight into the “modes-of-action” of nanoparticles and which of them may need to be further investigated for risk assessment.In 2008, a number of international researchers concluded that some published results of nanomaterial toxicity could not be replicated across laboratories and that accur
24、ate and reproducible nanotoxicology tests were needed. As a result of this, the International Alliance for NanoEHS Harmonization (IANH) was formed with the goal of developing testing protocols that would accurately assess toxicity and biological interactions of nanoparticles in cellular systems and
25、that these be reproducible in any laboratory. The IANH performed round robin characterization of particle size distributions in liquid suspensions and in vitro interactions of nanomaterials with cells with the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (
26、MTS), 5-(and-6)-chloromethyl-2,7 Dichloro-dihydro-fluorescein diacetate, acetyl ester (CM-H2DCF-DA), and propidium iodide assays. The IANH identified a number of factors that increased variability and developed techniques to reduce variability.Oxidative stress, which leads to DNA damage, is a primar
27、y driver leading to the accumulation of mutations which occurs in living organisms, so it is important to assess whether nanoparticles can induce reactive oxygen species in living cells.This document is a method to assess potential nanoparticle induced radical oxygen species (ROS) generation in cell
28、s through in vitro measurements. Although multiple techniques are used for determining generation of oxygen radicals in cells, the CM-H2DCF-DA has been used in round robin testing to evaluate ROS generation in mouse macrophages (RAW 264.7). The CM-DCF-DA assay provides a general measure of oxidative
29、 stress rather than detecting specific oxygen radicals or reactive species.4While this assay has not been evaluated in a broad range of cells, it does provide insight into the potential for ROS generation in macrophages which may play an important role in scavenging particles from the body.The CM-DC
30、F-DA assay has a margin of error even when controls are used and a number of factors could produce false negatives.4The CM-H2DCF-DA assay is not optimal for detecting all ROS species, such as the superoxide anion and hydroxyl radical which have short half-lives. In addition, measurement using cytome
31、try should be performed quickly after cells have been exposed in the assay, because DCFH and DCF can leach from cells or the DCFH can be oxidized. Also, the CM-H2DCF-DA assay is deactivated in serum, so cells should be washed to remove serum and cells could be lost in this process resulting in a pot
32、ential false negative. Furthermore, some nanoparticles may interact with DCFH and partially quench fluorescence. Thus, negative ROS results with this assay may not be conclusive. ISO/TS 18827 utilizes electron spin resonance (ESR) to detect the presence of ROS species in cells and differentiate betw
33、een the different reactive oxygen species without interference.In addition, there are several factors that could produce false-positive results.5Some nanoparticles and dead cells can fluoresce. Some nanoparticles can catalytically interact with CM-H2DCF-DA or the assay components can preferentially
34、adsorb on the surface of the particle.5In order to establish true positives, controls should be established to characterize nanoparticles alone under test conditions, as well as distinguish dead cell fluorescence from live cells with ROS.Furthermore, due to light-induced auto-oxidation, CM-H2DCF-DA
35、solutions at any concentration should be protected from light and air by storing in the dark in a sealed container filled with nitrogen gas or argon. ISO 2016 All rights reserved vPD ISO/TS 19006:2016ISO/TS 19006:2016(E)Thus, the CM-H2DCF-DA assay may be applicable to only particular cell lines and
36、nanoparticles and outcomes should be confirmed by additional assays (see Annex A for alternate cell lines). In particular, as a number of factors could lead to false negatives, or positives, other tests should be pursued and a positive result should be confirmed to not be caused by interference.Cont
37、rols are needed to determine a baseline of fluorescence of unexposed cells, determine whether cells are affected by non-toxic nanoparticles and also to demonstrate that known ROS generating chemicals and nanoparticles produced ROS which could be determined under assay conditions. Furthermore, it is
38、important to determine whether nanoparticles interfere with the fluorescence of the assay and potentially invalidate assessment of nanoparticle induced ROS generation in cells. Controlled experiments could be performed with cells exposed to Sin-1 with varying concentrations of nanoparticles present
39、to determine whether the nanoparticles quench the fluorescence.NOTE This assay is considered to be a screening assay that rapidly provides information about a nanoparticle interaction with a cellular system. Although screening type assays are critical for use in evaluating nanoparticle effects on ce
40、lls, it is important that interpretation of the results be verified with other ROS and related cellular assays.vi ISO 2016 All rights reservedPD ISO/TS 19006:2016TECHNICAL SPECIFICATION ISO/TS 19006:2016(E)Nanotechnologies 5-(and 6)-Chloromethyl-2,7 Dichloro-dihydrofluorescein diacetate (CM-H2DCF-DA
41、) assay for evaluating nanoparticle-induced intracellular reactive oxygen species (ROS) production in RAW 264.7 macrophage cell line1 ScopeThis document describes how to test and evaluate results obtained from in vitro ROS generation in RAW 264.7 macrophage cells exposed to nano-objects, nanoparticl
42、es, their aggregates and agglomerates using the CM-H2DCFDA assay.The protocol in this document is limited to use of a 24 well plate so if other plates were to be used, volumes would need to be adjusted and the protocol steps validated to ensure confidence in the test results.2 Normative referencesTh
43、ere are no normative references in this document.3 Terms and definitionsFor the purposes of this document, the terms and definitions given in ISO/TS 80004-2, ISO 10993-5 and the following apply.ISO and IEC maintain terminological databases for use in standardization at the following addresses: ISO O
44、nline browsing platform: available at http:/www.iso.org/obp IEC Electropedia: available at http:/www.electropedia.org/3.1agglomeratecollection of weakly bound particles or aggregates (3.2) or mixtures of the two where the resulting external surface area is similar to the sum of the surface areas of
45、the individual componentsNote 1 to entry: The forces holding an agglomerate together are weak forces (for example, van der Waals forces) or simple physical entanglement.Note 2 to entry: Agglomerates are also termed secondary particles and the original source particles are termed primary particles.SO
46、URCE: ISO/TS 80004-2:2015, 3.43.2aggregateparticle comprising strongly bonded or fused particles where the resulting external surface area may be significantly smaller than the sum of calculated surface areas of the individual componentsNote 1 to entry: The forces holding an aggregate together are s
47、trong forces (for example, covalent bonds) or those resulting from sintering or complex physical entanglement.Note 2 to entry: Aggregates are also termed secondary particles and the original source particles are termed primary particles. ISO 2016 All rights reserved 1PD ISO/TS 19006:2016ISO/TS 19006
48、:2016(E)SOURCE: ISO/TS 80004-2:2015, 3.53.3culture vesselsvessels appropriate for cell culture including glass Petri dishes, plastic culture flasks or plastic multiwall plates and microtitre platesNote 1 to entry: These can be used interchangeably in these methods provided that they meet the require
49、ments of tissue culture grade and are suitable for use with mammalian cells.SOURCE: ISO 10993-5:2009, 3.13.4dispersionmicroscopic multi-phase system in which discontinuities of any state (solid, liquid or gas: discontinuous phase) are dispersed in a continuous phase of a different composition or stateNote 1 to entry: If solid particles are dispersed in a liquid, the dispersion is referred to as a suspension. If the dispersion consists of two or more liquid phases, it is termed an emulsion. A suspoemulsion consis
