OxiSelect™ Intracellular ROS Assay Kit (Green Fluorescence)

Details for Product No. ABIN2344999, Supplier: Log in to see
Detection Method
Biochemical Assay (BCA)
Log in to see
Supplier Product No.
Log in to see
Purpose The OxiSelect™ Intracellular ROS Assay Kit is a cell-based assay for measuring antioxidant or ROS activity.
Brand OxiSelect™
Sample Type Cell Samples
Detection Method Fluorometric
Sensitivity 10 pM
Characteristics OxiSelect™ Intracellular ROS Assay Kit (Green Fluorescence) is a cell-based assay for measuring hydroxyl, peroxyl, or other reactive oxygen species activity within a cell. The assay employs the cell-permeable fluorogenic probe 2', 7'-Dichlorodihydrofluorescin diacetate (DCFH-DA). In brief, DCFH-DA is diffused into cells and is deacetylated by cellular esterases to non-fluorescent 2', 7'-Dichlorodihydrofluorescin (DCFH), which is rapidly oxidized to highly fluorescent 2', 7'- Dichlorodihydrofluorescein (DCF) by ROS (Figure 1). The fluorescence intensity is proportional to the ROS levels within the cell cytosol. The effect of antioxidant or free radical compounds on DCF- DA can be measured against the fluorescence of the provided DCF standard. The kit has a DCF detection sensitivity limit of 10 pM. Each kit provides sufficient reagents to perform up to 96 assays, including standard curve and unknown samples.
  1. 20X DCFH-DA : One 500 μL amber tube of a 20 mM solution in methanol.
  2. DCF Standard : One 100 μL amber tube of a 1 mM solution in DMSO.
  3. Hydrogen Peroxide : One 100 μL amber tube of an 8.821 M solution.
  4. 2X Cell Lysis Buffer : One 20 mL bottle. 3
Material not included
  1. Sterile DPBS for washes and buffer dilutions
  2. Hank's Balanced Salt Solution (HBSS)
  3. Cell culture medium (ie: DMEM +/-10 % FBS)
  4. 96-well black or fluorescence microtiter plate
  5. Fluorescent microplate reader capable of reading 480 nm (excitation) and 530 nm (emission)
Background Accumulation of reactive oxygen species (ROS) coupled with an increase in oxidative stress has been implicated in the pathogenesis of several disease states. The role of oxidative stress in vascular diseases, diabetes, renal ischemia, atherosclerosis, pulmonary pathological states, inflammatory diseases, and cancer has been well established. Free radicals and other reactive species are constantly generated in vivo and cause oxidative damage to biomolecules, a process held in check by the existence of multiple antioxidant and repair systems as well as the replacement of damaged nucleic acids, proteins and lipids. Measuring the effect of antioxidant therapies and ROS activity intracellularly is crucial to suppressing or treating oxidative stress inducers.
Application Notes Optimal working dilution should be determined by the investigator.

  • Quick ~1 hour protocol
  • Highly sensitive to 10 pM
  • Detects the presence of various ROS species

Assay Time 1 h
Protocol Cells are cultured in a 96-well cell culture plate and then pre-incubated with DCFH-DA, which is cell-permeable . The unknown antioxidant or ROS samples are then added to the cells. After a brief incubation, the cells can be read on a standard fluorescence plate reader. The ROS or antioxidant content in unknown samples is determined by comparison with the predetermined DCF standard curve.
Reagent Preparation
  • 1X DCFH-DA: Dilute the 20X DCFH-DA stock solution to 1X in cell culture media, preferably without FBS. Stir or vortex to homogeneity. Prepare only enough for immediate applications. Notes:
  • 1X DCFH-DA/media solution contains 5 % methanol. For cells that are sensitive to methanol, we recommend instead preparing a 0.1X (100 μM) solution of DCFH-DA in cell culture media.
  • Due to light-induced auto-oxidation, DCFH-DA solutions at any concentration must be protected from light.
  • Hydrogen Peroxide (H2O2): Prepare H2O2 dilutions in DMEM or DPBS as necessary. Do not store diluted solutions. Hydrogen Peroxide may be used as a positive control in the assay, or as a cell treatment.
Assay Procedure

I. DCF Dye Loading

  1. Prepare and mix all reagents thoroughly before use. Each unknown sample should be assayed in duplicate or triplicate.
  2. Culture cells in either a clear or black 96-well cell culture plate. Note: If using a black plate, choose an appropriate plate based on your fluorometer's reader (i.e. choose a clear bottom black plate for bottom readers).
  3. Remove media from all wells and discard. Wash cells gently with DPBS or HBSS 2-3 times. Remove the last wash and discard.
  4. Add 100 μL of 1X DCFH-DA/media solution to cells. Incubate at 37 °C for 30-60 minutes.
  5. Remove solution. Repeat step three using multiple washes with DPBS or HBSS. Remove the last wash and discard.
  6. Treat DCFH-DA loaded cells with desired oxidant or antioxidant in 100 μL medium. II. Quantitation of Fluorescence • Fluorescence microscopy or Flow cytometry: Fluorescence can be analyzed on an inverted fluorescence microscope or by flow cytometry using excitation and emission wavelengths of 480 nm and 530 nm, respectively. • Fluorescence Plate Reader: • Assays performed in black cell culture fluorometric plates: Plate may be read immediately for kinetic analysis or after 1 hour for static analysis. Plates read for kinetic analysis may be read in increments of 1 and 5 minutes up to 1 hour or more as necessary. Read the fluorescence with a fluorometric plate reader at 480 nm/530 nm. • Assays performed in clear cell culture plates: After treatment with desired oxidant or antioxidant, carefully remove treatment media from all wells and discard. Wash cells gently with DPBS or HBSS 2-3 times. Remove the last wash and discard. Add 100 μL of medium to each well. Add 100 μL of the 2X Cell Lysis Buffer, mix thoroughly and incubate 5 minutes. Transfer 150 μL of the mixture to a 96-well plate suitable for fluorescence measurement. Read the fluorescence with a fluorometric plate reader at 480 nm/530 nm.

Restrictions For Research Use only
Handling Advice Avoid multiple freeze/thaw cycles.
Storage 4 °C/-20 °C
Storage Comment Upon receipt, store the DCFH-DA and DCF Standard at -20°C. Avoid multiple freeze/thaw cycles. Store the Cell Lysis Buffer and Hydrogen Peroxide at 4°C.
Supplier Images
Cellular Assay (CA) image for OxiSelect™ Intracellular ROS Assay Kit (Green Fluorescence) (ABIN2344999) ROS in HeLa Cells Treated with Hydrogen Peroxide. 50,000 HeLa cells in a 96-well plat...
Product cited in: Yao, Zou, Wang, Ji, Yang: "Pinoresinol Diglucoside Alleviates oxLDL-Induced Dysfunction in Human Umbilical Vein Endothelial Cells." in: Evidence-based complementary and alternative medicine : eCAM, Vol. 2016, pp. 3124519, 2017 (PubMed).

Golestaneh, Chu, Xiao, Stoleru, Theos: "Dysfunctional autophagy in RPE, a contributing factor in age-related macular degeneration." in: Cell death & disease, Vol. 8, Issue 1, pp. e2537, 2017 (PubMed).

Crookenden, Walker, Heiser, Murray, Dukkipati, Kay, Meier, Moyes, Mitchell, Loor, Roche: "Effects of precalving body condition and prepartum feeding level on gene expression in circulating neutrophils." in: Journal of dairy science, Vol. 100, Issue 3, pp. 2310-2322, 2017 (PubMed).

Zhang, Zhao, Sun, Li, Wei, Ashman, Hu: "Different virulence of candida albicans is attributed to the ability of escape from neutrophil extracellular traps by secretion of DNase." in: American journal of translational research, Vol. 9, Issue 1, pp. 50-62, 2017 (PubMed).

Ludwig, Teran, Teichgraeber, Hilger: "Nanoparticle-based hyperthermia distinctly impacts production of ROS, expression of Ki-67, TOP2A, and TPX2, and induction of apoptosis in pancreatic cancer." in: International journal of nanomedicine, Vol. 12, pp. 1009-1018, 2017 (PubMed).

Vijayarathna, Oon, Chen, Kanwar, Sasidharan: "Polyalthia longifolia Methanolic Leaf Extracts (PLME) induce apoptosis, cell cycle arrest and mitochondrial potential depolarization by possibly modulating the redox status in hela cells." in: Biomedicine & pharmacotherapy, Vol. 89, pp. 499-514, 2017 (PubMed).

Kavitha, Ein Oon, Chen, Kanwar, Sasidharan: "Phaleria macrocarpa (Boerl.) fruit induce G0/G1 and G2/M cell cycle arrest and apoptosis through mitochondria-mediated pathway in MDA-MB-231 human breast cancer cell." in: Journal of ethnopharmacology, Vol. 201, pp. 42-55, 2017 (PubMed).

Xu, Kong: "Bixin ameliorates high fat diet-induced cardiac injury in mice through inflammation and oxidative stress suppression." in: Biomedicine & pharmacotherapy, Vol. 89, pp. 991-1004, 2017 (PubMed).

Yu, Pan, Dong, Niu: "Astragaloside IV attenuates lead acetate-induced inhibition of neurite outgrowth through activation of Akt-dependent Nrf2 pathway in vitro." in: Biochimica et biophysica acta, Vol. 1863, Issue 6, pp. 1195-1203, 2017 (PubMed).

Domey, Bergemann, Bremer-Streck, Krumbein, Reichenbach, Teichgräber, Hilger: "Long-term prevalence of NIRF-labeled magnetic nanoparticles for the diagnostic and intraoperative imaging of inflammation." in: Nanotoxicology, Vol. 10, Issue 1, pp. 20-31, 2016 (PubMed).

Wang, Sun, Chen, Jiao, Bai: "ROS-mediated activation of JNK/p38 contributes partially to the pro-apoptotic effect of ajoene on cells of lung adenocarcinoma." in: Tumour biology, 2016 (PubMed).

Slusarczyk, Trojan, Glombik, Piotrowska, Budziszewska, Kubera, Popiolek-Barczyk, Lason, Mika, Basta-Kaim: "Anti-inflammatory properties of tianeptine on lipopolysaccharide-induced changes in microglial cells involve toll-like receptor-related pathways." in: Journal of neurochemistry, Vol. 136, Issue 5, pp. 958-70, 2016 (PubMed).

Marondedze, Wong, Thomas, Irving, Gehring: "Cyclic Nucleotide Monophosphates in Plants and Plant Signaling." in: Handbook of experimental pharmacology, 2016 (PubMed).

Kuranaga, Yamada, Kashiwaya, Nakamura, Cui, Kumazaki, Shinohara, Sugito, Taniguchi, Ito, Nakayama, Uno, Itoh, Akao: "Anti-Oncogenic gem-Dihydroperoxides Induce Apoptosis in Cancer Cells by Trapping Reactive Oxygen Species." in: International journal of molecular sciences, Vol. 17, Issue 1, 2016 (PubMed).

Yamaguchi, Madhyastha, Madhyastha, Choijookhuu, Hishikawa, Pengjam, Nakajima, Maruyama: "Arsenic acid inhibits proliferation of skin fibroblasts, and increases cellular senescence through ROS mediated MST1-FOXO signaling pathway." in: The Journal of toxicological sciences, Vol. 41, Issue 1, pp. 105-13, 2016 (PubMed).

Johnson, Dludla, Joubert, February, Mazibuko, Ghoor, Muller, Louw: "Aspalathin, a dihydrochalcone C-glucoside, protects H9c2 cardiomyocytes against high glucose-induced shifts in substrate preference and apoptosis." in: Molecular nutrition & food research, 2016 (PubMed).

Liu, Chen, Xue, Zhao, Di: "Mefloquine effectively targets gastric cancer cells through phosphatase-dependent inhibition of PI3K/Akt/mTOR signaling pathway." in: Biochemical and biophysical research communications, Vol. 470, Issue 2, pp. 350-5, 2016 (PubMed).

Tu, Zhang, Wei, Li, Zhang, Yang, Xing: "Allicin attenuates H2O2?induced cytotoxicity in retinal pigmented epithelial cells by regulating the levels of reactive oxygen species." in: Molecular medicine reports, 2016 (PubMed).

Kim, Beaven, Kulinski, Desai, Bandara, Bai, Prussin, Schwartz, Komarow, Metcalfe, Olivera: "Regulation of Reactive Oxygen Species and the Antioxidant Protein DJ-1 in Mastocytosis." in: PLoS ONE, Vol. 11, Issue 9, pp. e0162831, 2016 (PubMed).

Subramani, Gonzalez, Arumugam, Nandy, Gonzalez, Medel, Camacho, Ortega, Bonkoungou, Narayan, Dwivedi, Lakshmanaswamy: "Nimbolide inhibits pancreatic cancer growth and metastasis through ROS-mediated apoptosis and inhibition of epithelial-to-mesenchymal transition." in: Scientific reports, Vol. 6, pp. 19819, 2016 (PubMed).