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This Technical Specification provides requirements for evaluation of the in vitro ROS generation in RAW 264.7
macrophage cells exposed to nanoparticles using the CM-H2DCFDA assay. In this test CM-H2DCF-DA
passively diffuses into cells, where its acetate groups are cleaved by intracellular esterases and its thiolreactive chloromethyl group reacts with intracellular glutathione and other thiols. Subsequent oxidation
yields a fluorescent adduct that is trapped inside the cell. This assay has been validated for polystyrene
nanoparticles, some metal nanoparticles, and their aggregates and agglomerates, and may be applicable
for other types of nanoobjects and their aggregates and agglomerates (NOAA).

Although parts of the protocol described in this document can be used with other cells and cell viability
assays, the protocol steps must be validated with the new cells and assays to ensure confidence in the
test result.

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.

This Technical Specification is intended to be used in biological testing laboratories that are competent in the culture and growth of cells and the use of flow cytometry.

This document is applicable to materials that consist of nano-objects such as nanoparticles, as well as
aggregates and agglomerates of these materials.

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.

This Technical Specification is a method to assess potential nanoparticle induced radical oxygen species(ROS) generation in cells through in vitro measurements.