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
ISO/TS 19006:2016 describes how to test 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-H2DCFDA assay.
The protocol in ISO/TS 19006:2016 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.
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Foreword
Introduction
1 Scope
2 Normative references
3 Terms and definitions
4 Symbols and abbreviated terms
5 Materials
6 Technical equipment
7 Nanoparticle sample preparation
8 Preparations
8.1 General
8.2 Flow cytometry calibration
8.3 Experimental culture medium
8.4 Reagent preparation
8.5 Preparation of cell stock culture
8.6 Preparing culture for experiments
8.7 Verification of healthy cell growth
8.8 Evaluation of nanoparticle interference
8.9 Control preparation
8.9.1 General
8.9.2 Control description
8.9.3 Sin-1 stock solution preparation (1 mM)
9 Evaluation of nanoparticle impact on ROS generation in cells
9.1 Prepare cells in the 24 well plates
9.2 Dose the cells with nanoparticles and controls
9.3 Expose the cells to CM-H2DCF-DA Assay
9.4 Incubate the cells with CM-H2DCF-DA
9.5 Flow cytometry analysis
10 Data analysis and results
Annex A (informative) Alternate cell lines
Annex B (informative) Alternate fluorescence characterization techniques
Annex C (informative) Suspension preparation and characterization
Annex D (informative) Example experimental data from RAW 264.7
Bibliography
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Foreword
Introduction
1 Scope
2 Normative references
3 Terms and definitions
4 Symbols and abbreviated terms
5 Materials
6 Technical equipment
7 Nanoparticle sample preparation
8 Preparations
8.1 General
8.2 Flow cytometry calibration
8.3 Experimental culture medium
8.4 Reagent preparation
8.5 Preparation of cell stock culture
8.6 Preparing culture for experiments
8.7 Verification of healthy cell growth
8.8 Evaluation of nanoparticle interference
8.9 Control preparation
8.9.1 General
8.9.2 Control description
8.9.3 Sin-1 stock solution preparation (1 mM)
9 Evaluation of nanoparticle impact on ROS generation in cells
9.1 Prepare cells in the 24 well plates
9.2 Dose the cells with nanoparticles and controls
9.3 Expose the cells to CM-H2DCF-DA Assay
9.4 Incubate the cells with CM-H2DCF-DA
9.5 Flow cytometry analysis
10 Data analysis and results
Annex A (informative) Alternate cell lines
Annex B (informative) Alternate fluorescence characterization techniques
Annex C (informative) Suspension preparation and characterization
Annex D (informative) Example experimental data from RAW 264.7
Bibliography