Amplex red h2o2 peroxidase assay kit

To detect ROS, treated RBCs were resuspended at 0.125% hematocrit in PBS and incubated with CM-H2-DCFDA (Invitrogen, Carlsbad, CA) at 8 μM for 30 min at 37 °C. Samples were then washed once with warm PBS and analyze for mean fluorescence intensity of 2′,7′-dichlorofluorescein using a FACSCalibur flow cytometer (Becton–Dickinson). For each sample, a minimum of 100 000 RBCs were acquired and the data were analyzed using CellQuest software (Becton–Dickinson).
Additionally, H₂O₂ was determined. The treated RBCs were incubated with fluorimetric horseradish peroxidase-catalyzed oxidation of Amplex Red using Amplex Red H₂O₂/Peroxidase Assay Kit (Molecular Probes, Grand Island, NY) according to the manufacturer's instructions. Absorbance was determined at 560 nm at multiple time points to follow the kinetics of the reactions.

The SD or control mice were anesthetized with ketamine/xylazine. To harvest tears, 2 μL of PBS was placed on the ocular surface of each eye. Following 10 s of mixture, 2 μL of irrigated tears were harvested by pipette and immediately diluted to 98 μL with PBS. For murine corneal epithelium sample collection, the whole corneal epithelial layer was removed by using a corneal rust ring remover (Algerbrush II, USA) and was immediately transferred into 100 μL of cold PBS. After homogenizing the tissue, the supernatant was collected for further detection. H₂O₂ concentration in tear and corneal epithelium samples was measured using the Amplex Red H₂O₂/peroxidase assay kit (Invitrogen, Carlsbad, CA). Total antioxidant capacity was determined using the FRAP antioxidant assay kit (Beyotime, Shanghai, China). The GSH and GPX were detected using the total GSH assay kit (Beyotime, Shanghai, China) and total GPX assay kit (Beyotime, Shanghai, China), respectively.

For in situ detection of H₂O₂, DAB was dissolved in deionized water and the pH of the solution was adjusted to pH 3.8 with HCl. Petals were infiltrated by vacuum in 1 mg mL⁻¹ DAB staining solution and were then incubated in the dark for 8 h. Stained petals were transferred to bleaching solution (ethanol:acetic acid:glycerol [3:1:1, v/v/v]) for 3 days. The DAB staining picture was converted to a grayscale image with Photoshop CS6 software, and image intensity was then determined with ImageJ software (Wu et al., 2015 (link)).
Determination of H₂O₂ concentration was performed using the Amplex Red H₂O₂/Peroxidase Assay Kit (Invitrogen), according to the manufacturer’s instructions with minor modifications. Briefly, petals were frozen in liquid nitrogen and ground into a fine powder; 30 mg of each sample was fully suspended in 200 μL H₂O₂ extraction buffer (25 mM sodium phosphate buffer, pH 6.5). The extracts were centrifuged at 13,000 rpm for 15 min at 4°C, and the supernatant was prepared for quantification (Guo et al., 2017 (link)). Fluorescence was then measured with a Thermo Scientific Varioskan Flash reader using excitation at 530 nm and fluorescence detection at 590 nm.