Amplex red hydrogen peroxide peroxidase assay kit

The total level of ROS were measured by using either a small diffusible fluorescent probe (chloromethyl derivative; CM-H₂DCFDA; Life Technologies) or with the Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit (Life Technologies) with a protocol adapted from Schulz et al. (2007) (link). Briefly, wild-type eggs were hatched on empty vector (EV) or memo-1(RNAi) food and harvested for assay at larval stage 4 (L4). More than 1000 L4 animals per conditions were harvested into 96-well plates, incubated in 50 µM CM-H₂DCFDA for 1 hr and the fluorescent intensity was measured with an excitation wavelength of 485 nm and a 520 nm emission filter. Detection of ROS by Amplex Red was performed according to manufacturer’s instructions and fluorescence was measured with an excitation wavelength of 530 nm and a 590 nm emission filter. Animals were lysed and protein levels were determined using BCA assay (Pierce). The fluorescent intensity was normalized to protein levels and is indicated relative to control.

Targets. pHrodo™ Green E. coli BioParticles™ (Cat # P35366), Zymosan A (S. cerevisiae) BioParticles™, Alexa Fluor™ 488 conjugated (Cat # Z23373), E. coli BioParticles™ Opsonizing Reagent (Cat # E2870), Zymosan A BioParticles™ Opsonizing Reagent (Cat # Z2850), and Amplex™ Red Hydrogen Peroxide/Peroxidase Assay Kit (Cat # A22188) were purchased from Life Technologies. E. coli and Zymosan were IgG-opsonized per manufacturer's instructions.

In this study, five Cerium Oxide Nanoparticles (CNPs) were synthesized using different methods with varying % surface Ce³⁺, size and morphology. 99.999% pure cerium nitrate hexahydrate was used for all the preparation. CNP1 and CNP2 were synthesized using the wet chemical method, with H₂O₂ (CNP1) and NH₄OH (CNP2) as oxidizing agents45 (link). In order to prove that there was no H₂O₂ in CNP1 after the synthesis process, the Amplex® Red Hydrogen Peroxide/Peroxidase Assay Kit was performed following the manufacturer instructions (Life Technologies). Briefly, reactions containing 50 μM Amplex® Red reagent, 0.1 U/mL horseradish peroxidase (HRP) and increasing concentration of CNP1 (1, 10, 100 mg/l) were incubated for 30 minutes at room temperature. Fluorescence was then measured with a Fluorostar Omega plate reader (BMG LABTECH GmbH, Germany) using excitation at 530 nm and fluorescence detection at 590 nm. H₂O₂ was not detected in any of the concentration tested (see Supplementary Figure S3). No differences were found between CNP1 and control (with H₂O). CNP3, CNP4 and CNP5 were prepared using the hydrothermal method, as described elsewhere44 . The % surface Ce³⁺ for all the nanoparticles were tested several times over the experimental period in the aqueous environment (data not shown) and the % surface Ce³⁺/Ce⁴⁺ was stable for all these nanoparticles.

Intracellular ROS were detected by CM-H₂DCFDA (Life technologies, C6827). Liver non-parenchymal cells were stained with 5 μM H₂DCFDA in 37 °C incubator for 30 min followed by washing twice in PBS before FACS analysis. The concentration of Hydrogen peroxide was measured by an Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit (Life Technologies, A22188).
Calcium influx was determined using a Fluo-4 Direct Calcium Assay Kit (Thermo Fisher, F10471). Fluo-4 was recorded over time on a BD FACSVerse flow cytometer.