ISO/TS TECHNICAL SPECIFICATION 19006 Firstedition 2016-08-01 Nanotechnologies 5 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-Essaiaudiacetatede5-(et6)-Chlorométhyle -2'7'Dichloro-dihydro-fluoresceine (CM-H2DCF-DA)pour Il'evaluation de la generation intracellulaire d'especes reactives a I'oxygene induitespar les nanoparticules sur la lignee souche264.7de macrophages Reference number IS0/TS19006:2016(E) ISO Intermational Organization for Standardization @ISO2016 IS0/TS 19006:2016(E) COPYRIGHTPROTECTEDDOCUMENT IS02016,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, including 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 ISO's member body in the country of the requester. ISOcopyrightoffice Ch.de Blandonnet 8.CP 401 CH-1214 Vernier, Geneva, Switzerland Tel.+4122 749 0111 Fax+41227490947 [email protected] www.iso.org No reproduction or networking permited withoutlicense from IHS IS0/TS19006:2016(E) Contents Page Foreword ..iv Introduction. ..V 1 Scope. 2 Normative references 3 Terms and definitions ..1 4 Symbols and abbreviated terms ..3 5 Materials ..3 6 Technical equipment 7 Nanoparticle samplepreparation .5 8 Preparations .6 8.1 General. .6 8.2 Flowcytometrycalibration 7 8.3 Experimental culture medium .7 8.4 Reagentpreparation. .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 Evaluationofnanoparticleinterference .9 8.9 Control preparation. .9 8.9.1 General .9 8.9.2 Control description. 9 8.9.3 Sin-1stocksolutionpreparation (1mM) .10 9 Evaluation ofnanoparticleimpact on RoSgeneration in cells .10 9.1 Preparecellsinthe24wellplates. .10 9.2 Dosethe cells with nanoparticles and controls ..10 9.3 Expose the cells to CM-H2DCF-DA Assay ..11 9.4 IncubatethecellswithCM-H2DCF-DA .12 9.5 Flowcytometryanalysis ..12 10 Data analysis and results ..12 AnnexA(informative)Alternatecelllines. ..14 AnnexB(informative)Alternatefluorescencecharacterizationtechniques ..15 AnnexC (informative)Suspensionpreparationandcharacterization ..16 AnnexD(informative)ExampleexperimentaldatafromRAW264.7 ..17 Bibliography .20 iii thout license from IHS