OxiSelect™ In Vitro ROS/RNS Assay Kit (Green Fluorescence)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
		- Due to light-induced auto-oxidation, the stock DCF-DiOxyQ solution and all subsequent DCF-DiOxy and DCFH solutions must be protected from light.

Downstream tips
- Due to light-induced auto-oxidation, the stock DCF-DiOxyQ solution and all subsequent DCF-DiOxy and DCFH solutions must be protected from light.

Publication protocol

Fibroblasts from patients with mitochondrial diseases were cultured in 1.0 g/L low-glucose Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% FBS, 100 units/ml penicillin and 100 µg/ml streptomycin (Normal growth medium) at 37°C, 95% ambient air and 5% CO2 and 21% oxygen for maintaining culture. In cell viability assay, low-glucose DMEM with 1% FBS and antibiotics (assay medium) was used. Mitochondrial disease fibroblasts were cultured in normal growth medium until semi-confluent and then plating at 1.5 × 104 cells/ 0.5 ml assay medium per each well of 48 well flat bottom culture plate in assay medium. After 24-hr incubation, L-buthionine- (S,R)-sulfoximine (BSO) at 100 µM or control vehicle dimethyl sulfoxide (DMSO) at 0.1% were added and cultured for another 24 hrs. After each compound was applied at indicated final concentrations, fibroblasts were cultured for 48 hrs, and cell viability was measured by Cell count Regent SF (nacalai tesque) (Enns et al. 2012). Intracellular ATP was measured with Cellno ATP assay regent (TOYO B-NET). Reactive oxygen species (ROS) were measured by OxiSelect ROS Assay Kit (Cell Biolabs). Glutathione (GSH) was measured using a GSH-Glo™ Glutathione Assay kit based on the manufacturer’s instructions (Promega).

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Papers

Check out relevant papers found by Labettor's AI that are relevant for performing ROS assay cell type - PLHC-1, SK-HEP-1, Hep3b, HepG2 human hepatocellular carcinoma using OxiSelect™ In Vitro ROS/RNS Assay Kit (Green Fluorescence) from Cell Biolabs.

Manufacturer protocol

Download the product protocol from Cell Biolabs for OxiSelect™ In Vitro ROS/RNS Assay Kit (Green Fluorescence) below.

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Videos

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