1、PD ISO/TS 19006:2016 Nanotechnologies 5-(and 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 line BSI Standards Publication WB11885_BSI_StandardCov
2、s_2013_AW.indd 1 15/05/2013 15:06PD ISO/TS 19006:2016 PUBLISHED DOCUMENT National foreword This Published Document is the UK implementation of ISO/TS 19006:2016. The UK participation in its preparation was entrusted to Technical Committee NTI/1, Nanotechnologies. A list of organizations represented
3、on this committee can be obtained on request to its secretary. This publication does not purport to include all the necessary provisions of a contract. Users are responsible for its correct application. The British Standards Institution 2016. Published by BSI Standards Limited 2016 ISBN 978 0 580 83
4、224 6 ICS 07.030 Compliance with a British Standard cannot confer immunity from legal obligations. This Published Document was published under the authority of the Standards Policy and Strategy Committee on 31 August 2016. Amendments issued since publication Date Text affectedPD ISO/TS 19006:2016 IS
5、O 2016 Nanotechnologies 5-(and 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 line Nanotechnologies Essai au diactate de 5-(et 6)- Chloromthyle -2,
6、7 Dichloro-dihydro-fluorescine (CM-H2DCF-DA) pour lvaluation de la gnration intracellulaire despces ractives loxygne induites par les nanoparticules sur la ligne souche 264.7 de macrophages TECHNICAL SPECIFICATION ISO/TS 19006 Reference number ISO/TS 19006:2016(E) First edition 2016-08-01PD ISO/TS 1
7、9006:2016ISO/TS 19006:2016(E)ii ISO 2016 All rights reserved COPYRIGHT PROTECTED DOCUMENT ISO 2016, Published in Switzerland All 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, inc
8、luding photocopying, or posting on the internet 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 office Ch. de Blandonnet 8 CP 401 CH-1214 Vernier, Geneva, Switzerland
9、 Tel. +41 22 749 01 11 Fax +41 22 749 09 47 copyrightiso.org www.iso.orgPD ISO/TS 19006:2016ISO/TS 19006:2016(E)Foreword iv Introduction v 1 Scope . 1 2 Normative references 1 3 Terms and definitions . 1 4 Symbols and abbreviated terms . 3 5 Materials . 3 6 Technical equipment 5 7 Nanoparticle sampl
10、e preparation 5 8 Preparations . 6 8.1 General . 6 8.2 Flow cytometry calibration 7 8.3 Experimental culture medium 7 8.4 Reagent preparation. 7 8.5 Preparation of cell stock culture 7 8.6 Preparing culture for experiments 7 8.7 Verification of healthy cell growth . 8 8.8 Evaluation of nanoparticle
11、interference . 9 8.9 Control preparation 9 8.9.1 General 9 8.9.2 Control description 9 8.9.3 Sin-1 stock solution preparation (1 mM) 10 9 Evaluation of nanoparticle impact on ROS generation in cells .10 9.1 Prepare cells in the 24 well plates . 10 9.2 Dose the cells with nanoparticles and controls .
12、10 9.3 Expose the cells to CM-H 2 DCF-DA Assay .11 9.4 Incubate the cells with CM-H 2 DCF-DA 12 9.5 Flow cytometry analysis 12 10 Data analysis and results 12 Annex A (informative) Alternate cell lines 14 Annex B (informative) Alternate fluorescence characterization techniques 15 Annex C (informativ
13、e) Suspension preparation and characterization 16 Annex D (informative) Example experimental data from RAW 264.7 17 Bibliography .20 ISO 2016 All rights reserved iii Contents PagePD ISO/TS 19006:2016ISO/TS 19006:2016(E) Foreword ISO (the International Organization for Standardization) is a worldwide
14、 federation of national standards bodies (ISO member bodies). The work of preparing International 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
15、committee. International organizations, governmental and non-governmental, in liaison with 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 docume
16、nt and those intended for its further maintenance are described in the ISO/IEC Directives, 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
17、, Part 2 (see www.iso.org/directives). Attention is drawn to the possibility that some of the 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
18、 of the document will be in the Introduction and/or on the ISO list of patent declarations 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 speci
19、fic terms and expressions related to conformit y assessment, as well as information about ISOs 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/
20、TC 229, Nanotechnologies.iv ISO 2016 All rights reservedPD ISO/TS 19006:2016ISO/TS 19006:2016(E) Introduction The 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 th
21、e potential impact on human health and on the environment of some of these materials. Internationally, 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
22、the interactions of nanoparticles with cells, even more so due to their large surface to volume ratio. 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 li
23、kely that monitoring biological response of cellular model systems to nanoparticle exposure can provide 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 pu
24、blished results of nanomaterial toxicity could not be replicated across laboratories and that accurate 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 wou
25、ld accurately assess toxicity and biological interactions of nanoparticles in cellular systems and 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
26、with the 3-(4,5-dimethylthiazol-2-yl)- 5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), 5-(and-6)-chloromethyl-2,7 Dichloro-dihydro-fluorescein diacetate, acetyl ester (CM-H 2 DCF-DA), and propidium iodide assays. The IANH identified a number of factors that increased variability a
27、nd developed techniques to reduce variability. Oxidative stress, which leads to DNA damage, is a primary 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 docum
28、ent is a method to assess potential nanoparticle induced radical oxygen species (ROS) generation in cells through in vitro measurements. Although multiple techniques are used for determining generation of oxygen radicals in cells, the CM-H 2 DCF-DA has been used in round robin testing to evaluate RO
29、S generation in mouse macrophages (RAW 264.7). The CM-DCF-DA assay provides a general measure of oxidative 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
30、 generation in macrophages which may play an important role in scavenging particles from the body. The CM-DCF-DA assay has a margin of error even when controls are used and a number of factors could produce false negatives. 4The CM-H 2 DCF-DA assay is not optimal for detecting all ROS species, such
31、as the superoxide anion and hydroxyl radical which have short half-lives. In addition, measurement using cytometry 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-H 2 DCF-DA assay is deactivat
32、ed in serum, so cells should be washed to remove serum and cells could be lost in this process resulting in a potential 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 1
33、8827 utilizes electron spin resonance (ESR) to detect the presence of ROS species in cells and differentiate between 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 fl
34、uoresce. Some nanoparticles can catalytically interact with CM-H 2 DCF-DA or the assay components can preferentially 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 dist
35、inguish dead cell fluorescence from live cells with ROS. Furthermore, due to light-induced auto-oxidation, CM-H 2 DCF-DA 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
36、vPD ISO/TS 19006:2016ISO/TS 19006:2016(E) Thus, the CM-H 2 DCF-DA assay may be applicable to only particular cell lines and 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,
37、 or positives, other tests should be pursued and a positive result should be confirmed to not be caused by interference. Controls 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
38、ROS generating chemicals and nanoparticles produced ROS which could be determined under assay conditions. Furthermore, it is important to determine whether nanoparticles interfere with the fluorescence of the assay and potentially invalidate assessment of nanoparticle induced ROS generation in cells
39、. Controlled experiments could be performed with cells exposed to Sin-1 with varying concentrations of nanoparticles present 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 in
40、teraction with a cellular system. Although screening type assays are critical for use in evaluating nanoparticle effects on cells, 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 SP
41、ECIFICATION ISO/TS 19006:2016(E) Nanotechnologies 5-(and 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 line 1 Scope This document describes how to t
42、est and evaluate results obtained from in vitro ROS generation in RAW 264.7 macrophage cells exposed to nano-objects, nanoparticles, their aggregates and agglomerates using the CM-H 2 DCFDA assay. The protocol in this document is limited to use of a 24 well plate so if other plates were to be used,
43、volumes would need to be adjusted and the protocol steps validated to ensure confidence in the test results. 2 Normative references There are no normative references in this document. 3 Terms and definitions For the purposes of this document, the terms and definitions given in ISO/TS 80004-2, ISO 10
44、993-5 and the following apply. ISO and IEC maintain terminological databases for use in standardization at the following addresses: ISO Online browsing platform: available at http:/ /www.iso.org/obp IEC Electropedia: available at http:/ /www.electropedia.org/ 3.1 agglomerate collection of weakly bou
45、nd 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 the individual components Note 1 to entry: The forces holding an agglomerate together are weak forces (for example, van der Waals forces) or simple physical
46、 entanglement. Note 2 to entry: Agglomerates are also termed secondary particles and the original source particles are termed primary particles. SOURCE: ISO/TS 80004-2:2015, 3.4 3.2 aggregate particle comprising strongly bonded or fused particles where the resulting external surface area may be sign
47、ificantly smaller than the sum of calculated surface areas of the individual components Note 1 to entry: The forces holding an aggregate together are strong forces (for example, covalent bonds) or those resulting from sintering or complex physical entanglement. Note 2 to entry: Aggregates are also t
48、ermed secondary particles and the original source particles are termed primary particles. ISO 2016 All rights reserved 1PD ISO/TS 19006:2016ISO/TS 19006:2016(E) SOURCE: ISO/TS 80004-2:2015, 3.5 3.3 culture vessels vessels appropriate for cell culture including glass Petri dishes, plastic culture fla
49、sks or plastic multiwall plates and microtitre plates Note 1 to entry: These can be used interchangeably in these methods provided that they meet the requirements of tissue culture grade and are suitable for use with mammalian cells. SOURCE: ISO 10993-5:2009, 3.1 3.4 dispersion microscopic 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 state Note 1 to entry: If solid particles are dispersed i
