Highly sensitive and specific fluorogenic probes to measure hypoxia and oxidative stress in live cells
Used with adherent or suspension cell lines
Complete set of reagents, including ROS and Hypoxia inducers
Enzo Life Sciences’ ROS-ID® Hypoxia/Oxidative stress detection kit is designed for functional detection of hypoxia and oxidative stress levels in live cells (both suspension and adherent) using fluorescent microscopy or flow cytometry. This kit includes fluorogenic probes for hypoxia (red) and for oxidative stress levels (green) as two major components.
The Hypoxia (Red) dye takes advantage of the nitroreductase activity present in hypoxic cells by converting the Nitro group to hydroxylamine (NHOH) and amino (NH2) and releasing the fluorescent probe.
The Oxidative Stress Detection Reagent is a non-fluorescent, cell-permeable total ROS detection dye which reacts directly with a wide range of reactive species. The generated fluorescent products can be visualized using a wide-field fluorescence microscope equipped with standard fluorescein (490/525 nm) and Texas Red (596/670 nm) filters, confocal microscopy, or cytometrically using any flow cytometer equipped with a blue (488 nm) laser.
Detection of hypoxia and oxidative stress levels in cultured human HeLa and HL-60 cells. Cells were treated with hypoxia inducer (DFO) and ROS inducer (pyocyanin). Numbers in each quadrant reflects the percentage of cells (population). Results indicate that hypoxia and oxidative stress dye are specific
HeLa cells were subject to treatment. Bright red fluorescence of the Hypoxia probe is observed following its conversion by cellular nitroreductases under hypoxic conditions such as those induced chemically by treatment with the hypoxia-mimetic desferrioxamine (DFO).
The absorption and emission peaks for the Oxidative Stress (A) and Hypoxia Red (B) detection dyes are 504nm/524nm and 580nm/595nm, respectively. The dyes can be excited with an argon ion laser at 488 nm, and detected in the FL1 channel (Oxidative Stress dye) and FL3 Channel (Hypoxia Red dye) on ost bench flow cytometers.
This kit is designed for fluorescence microscopy and/or flow cytometry using adherent or suspension cells.
Quality Control:
The testing is accomplished using flow cytometry method for assessment of hypoxic cells and/or cells with high levels of total oxidative stress in conjunction with dyes (provided in kit). Microscopy images are also obtained.
Quantity:
For -K500 size:
500 fluorescence microscopy assays or 100 flow cytometry assays.
For -0125 size:
125 fluorescence microscopy assays or 25 flow cytometry assays.
Use/Stability:
With proper storage, the kit components are stable up to the date noted on the product label.
The ROS-ID® Hypoxia/Oxidative stress detection kit is a member of the CELLESTIAL® product line, reagents and assay kits comprising fluorescent molecular probes that have been extensively benchmarked for live cell analysis applications. CELLESTIAL® reagents and kits are optimal for use in demanding cell analysis applications involving confocal microscopy, flow cytometry, microplate readers and HCS/HTS, where consistency and reproducibility are required.
Detailed instructions are included in the manual for microscopy and flow cytometry applications for adherent and suspension cells.
Regulatory Status:
RUO - Research Use Only
Product Literature References
Anoxia Rapidly Induces Changes in Expression of a Large and Diverse Set of Genes in Endothelial Cells: A. Antonelli, et al.; Int. J. Mol. Sci. 24, 5157 (2023), Abstract;
Cupric-ion-promoted fabrication of oxygen-replenishing nanotherapeutics for synergistic chemo and photodynamic therapy against tumor hypoxia: L. He, et al.; Acta Biomater. 162, 57 (2023), Abstract;
Precise manipulation of circadian clock using MnO2 nanocapsules to amplify photodynamic therapy for osteosarcoma: Y.X. Ge, et al.; Mater. Today Bio 19, 100547 (2023), Abstract;
The impact of hypoxia and oxidative stress on proteo-metabolomic alterations of 3D cholangiocarcinoma models: P. Phukhum, et al.; Sci. Rep. 13, 3072 (2023), Abstract;
Dimethyloxalylglycine (DMOG), a Hypoxia Mimetic Agent, Does Not Replicate a Rat Pheochromocytoma (PC12) Cell Biological Response to Reduced Oxygen Culture: R. Chen, et al.; Biomolecules 12, 541 (2022), Abstract;
Hydrogel microcapsules containing engineered bacteria for sustained production and release of protein drugs: C. Han, et al.; Biomaterials 287, 121619 (2022), Abstract;
Inhibiting autophagy flux and DNA repair of tumor cells to boost radiotherapy of orthotopic glioblastoma: Q. Xu, et al.; Biomaterials 280, 121287 (2022), Abstract;
Intracellular glucose starvation affects gingival homeostasis and autophagy: R. Li, et al.; Sci. Rep. 12, 1230 (2022), Abstract;
Intrinsic radical species scavenging activities of tea polyphenols nanoparticles block pyroptosis in endotoxin-induced sepsis: Y. Chen, et al.; ACS Nano 16, 2429 (2022), Abstract;
Iodinated cyanine dye-based nanosystem for synergistic phototherapy and hypoxia-activated bioreductive therapy: Y. Dong, et al.; Drug Deliv. 29, 238 (2022), Abstract;
Lipoprotein-biomimetic nanostructure enables tumor-targeted penetration delivery for enhanced photo-gene therapy towards glioma: R. Wang, et al.; Bioact. Mater. 13, 286 (2022), Abstract;
Microenvironment-driven sequential ferroptosis, photodynamic therapy, and chemotherapy for targeted breast cancer therapy by a cancer-cell-membrane-coated nanoscale metal-organic framework: W.L. Pan ,et al.; Biomaterials 283, 121559 (2022), Abstract;
Mitochondrial glutathione depletion nanoshuttles for oxygen-irrelevant free radicals generation: A cascaded hierarchical targeting and theranostic strategy against hypoxic tumor: B. Liang, et al.; ACS Appl. Mater. Interfaces 14, 13038 (2022), Abstract;
Multifunctional Nanosnowflakes for T1-T2 Double-Contrast Enhanced MRI and PAI Guided Oxygen Self-Supplementing Effective Anti-Tumor Therapy: Y. Lv, et al.; Int. J. Nanomedicine 17, 4619 (2022), Abstract;
Physiologic flow-conditioning limits vascular dysfunction in engineered human capillaries: K. Haase, et al.; Biomaterials 280, 121248 (2022), Abstract;
Platinum prodrug nanoparticles inhibiting tumor recurrence and metastasis by concurrent chemoradiotherapy: W. Jiang, et al.; J. Nanobiotechnology 20, 129 (2022), Abstract;
Strategy for improving cell-mediated vascularized soft tissue formation in a hydrogen peroxide-triggered chemically-crosslinked hydrogel: S.Y. Wei, et al.; J. Tissue. Eng. 13, 20417314221084096 (2022), Abstract; Full Text
A cyclic nano-reactor achieving enhanced photodynamic tumor therapy by reversing multiple resistances: P. Liu, et al.; J. Nanobiotechnology 19, 149 (2021), Abstract; Full Text
An albumin-based therapeutic nanosystem for photosensitizer/protein co-delivery to realize synergistic cancer therapy: S.L. Ai, et al.; ACS Appl. Bio. Mater. 4, 4946 (2021), Abstract;
An injectable hydrogel co-loading with cyanobacteria and upconversion nanoparticles for enhanced photodynamic tumor therapy: X. Zhang, et al.; Colloids Surf. B. Biointerfaces 201, 111640 (2021), Abstract;
Biomimetic nanoreactor for targeted cancer starvation therapy and cascade amplificated chemotherapy: F. Shao, et al.; Biomaterials 274, 120869 (2021), Abstract;
Bone-targeted erythrocyte-cancer hybrid membrane-camouflaged nanoparticles for enhancing photothermal and hypoxia-activated chemotherapy of bone invasion by OSCC: H. Chen, et al.; J. Nanobiotechnology 19, 342 (2021), Abstract; Full Text
Combination of coumarin and doxorubicin induces drug-resistant acute myeloid leukemia cell death: N.S. Al-Abbas, et al.; Heliyon 7, e06255 (2021), Abstract; Full Text
Encapsulating an acid-activatable phthalocyanine–doxorubicin conjugate and the hypoxia-sensitive tirapazamine in polymeric micelles for multimodal cancer therapy: X. Guo, et al.; Biomater. Sci. 9, 4936 (2021), Abstract;
Exploiting the acquired vulnerability of cisplatin-resistant tumors with a hypoxia-amplifying DNA repair-inhibiting (HYDRI) nanomedicine: J. Chen, et al.; Sci. Adv. 7, 5267 (2021), Abstract;
Extracellular matrix-induced GM-CSF and hypoxia Promote Immune Control of Mycobacterium tuberculosis in Human In Vitro Granulomas: A. Arbues, et al.; Front. Immunol. 12, 727508 (2021), Abstract; Full Text
Immune/Hypoxic Tumor Microenvironment Regulation-Enhanced Photodynamic Treatment Realized by pH-Responsive Phase Transition-Targeting Nanobubbles: M. Zhao, et al.; ACS Appl. Mater. Interfaces 13, 32763 (2021), Abstract;
Mitochondria-targeted and ultrasound-responsive nanoparticles for oxygen and nitric oxide codelivery to reverse immunosuppression and enhance sonodynamic therapy for immune activation: C. Ji, et al.; Theranostics 11, 8587 (2021), Abstract; Full Text
MnO2-based nanomotors with active fenton-like Mn2+ delivery for enhanced chemodynamic therapy: J. Ou, et al.; ACS Appl. Mater. Interfaces 13, 38050 (2021), Abstract;
Mucin 1 regulates the hypoxia response in head and neck cancer cells: K. Utispan, et al.; J. Pharmacol. Sci. 147, 331 (2021), Abstract;
Ruthenium (II) complexes coordinated to graphitic carbon nitride: Oxygen self-sufficient photosensitizers which produce multiple ROS for photodynamic therapy in hypoxia: F. Wei, et al.; Biomaterials 276, 121064 (2021), Abstract;
Stratified 3D microtumors as organotypic testing platforms for screening pancreatic cancer therapies: M.V. Monteiro, et al.; Small Methods 5, e2001207 (2021), Abstract;
Synergy of hypoxia relief and heat shock protein inhibition for phototherapy enhancement: G. Zhang, et al.; J. Nanobiotechnology 19, 9 (2021), Abstract; Full Text
The New Serum-Free OptiPASS ® Medium in Cold and Oxygen-Free Conditions: An Innovative Conservation Method for the Preservation of MDA-MB-231 Triple Negative Breast Cancer Spheroids: A. Goisnard, et al.; Cancers 13, 1945 (2021), Abstract; Full Text
Biomimetic decoy inhibits tumor growth and lung metastasis by reversing the drawbacks of sonodynamic therapy: H. Zhao, et al.; Adv. Healthc. Mater. 9, e1901335 (2020), Application(s): Fluorescence microscopy using 4T1 cells, Abstract;
Remodeling extracellular matrix based on functional covalent organic framework to enhance tumor photodynamic therapy: S.B. Wang, et al.; Biomaterials 234, 119772 (2020), Application(s): Fluorescence microscopy using CT26 cells, Abstract;
A two-photon excited O2-evolving nanocomposite for efficient photodynamic therapy against hypoxic tumor: R.Q. Li, et al.; Biomaterials 194, 84 (2019), Application(s): Fluorescence microscopy using 4T1 cells, Abstract;
Combinational phototherapy and hypoxia-activated chemotherapy favoring antitumor immune responses: B. Ma, et al.; Int. J. Nanomedicine 14, 4541 (2019), Application(s): Fluorescence microscopy using 4T1 cells, Abstract; Full Text
Di-(2-ethylhexyl) phthalate (DEHP) inhibits steroidogenesis and induces mitochondria-ROS mediated apoptosis in rat ovarian granulosa cells: A. Tripathi, et al.; Toxicol. Res. (Camb.) 8, 381 (2019), Application(s): Flow cytometry using granulosa cells, Abstract;
Encircling granulosa cells protects against di-(2-ethylhexyl)phthalate-induced apoptosis in rat oocytes cultured in vitro: A. Tripathi, et al.; Zygote 27, 203 (2019), Abstract;
Endogenous oxygen generating multifunctional theranostic nanoplatform for enhanced photodynamic-photothermal therapy and multimodal imaging: K. Wu, et al.; Theranostics 9, 7697 (2019), Application(s): Confocal microscopy using HeLa cells, Abstract; Full Text
Glutathione depletion and dual-model oxygen balance disruption for photodynamic therapy enhancement: W. Li, et al.; Colloids Surf. B Biointerfaces 183, 110453 (2019), Application(s): Fluorescence microscopy using 4T1 cells, Abstract;
Investigation of PPIX-Lipo-MnO2 to enhance photodynamic therapy by improving tumor hypoxia: L. Chudal, et al.; Mater. Sci. Eng. C Mater. Biol. Appl. 104, 109979 (2019), Application(s): Fluorescence microscopy using MCF-7 cells, Abstract;
Laser-triggered polymeric lipoproteins for precision tumor penetrating theranostics: R. Wang, et al.; Biomaterials 221, 119413 (2019), Application(s): Fluorescence microscopy using 4T1 cells, Abstract;
Monodispersed copper (I)‐based nano metal–organic framework as a biodegradable drug carrier with enhanced photodynamic therapy efficacy: X. Cai, et al.; Adv. Sci. 6, 1900848 (2019), Application(s): Fluorescence microscopy using 4T1 cells, Abstract; Full Text
Oral ferroportin inhibitor ameliorates ineffective erythropoiesis in a model of β-thalassemia: V. Manolova, et al.; J. Clin. Invest. 130, 491 (2019), Application(s): Flow cytometry analysis using mouse red blood cells, Abstract;
Oxygen-supplementing mesoporous polydopamine nanosponges with WS2 QDs-embedded for CT/MSOT/MR imaging and thermoradiotherapy of hypoxic cancer: Y. Wang, et al.; Biomaterials 220, 119405 (2019), Application(s): Fluorescence microscopy using 4T1 cells, Abstract;
Self-generating oxygen enhanced mitochondrion-targeted photodynamic therapy for tumor treatment with hypoxia scavenging: Z. Yang, et al.; Theranostics 9, 6809 (2019), Application(s): Fluorescence microscopy using MKN-45P cells, Abstract; Full Text
Solid matrix-supported supercritical CO2 enhances extraction of γ-linolenic acid from the cyanobacterium Arthrospira (Spirulina) platensis and bioactivity evaluation: X. Yang, et al.; Mar. Drugs 17, 203 (2019), Application(s): Fluorescence microscopy using Zebrafish larvae, Abstract; Full Text
All-in-one theranostic nanoplatform based on hollow MoSx for photothermally-maneuvered oxygen self-enriched photodynamic therapy: J. Wang, et al.; Theranostics 8, 955 (2018), Application(s): Fluorescence microscopy using 4T1 cells, Abstract; Full Text
Fluorinated polymeric micelles to overcome hypoxia and enhance photodynamic cancer therapy: Q. Wang, et al.; Biomater. Sci. 6, 3096 (2018), Abstract;
High glucose-induced oxidative stress impairs proliferation and migration of human gingival fibroblasts: P. Buranasin, et al.; PLoS One 13, e0201855 (2018), Abstract; Full Text
Light-enhanced hypoxia-responsive nanoparticles for deep tumor penetration and combined chemo-photodynamic therapy: Z. Li, et al.; Chem. Commun. 54, 13127 (2018), Abstract;
LSD1 mediated changes in the local redox environment during the DNA damage response: M.L. Duquette, et al.; PLoS One 13, e0201907 (2018), Abstract; Full Text
Platelet-mimicking nanoparticles co-loaded with W18O49 and metformin alleviate tumor hypoxia for enhanced photodynamic therapy and photothermal therapy: H. Zuo, et al.; Acta Biomater. 80, 296 (2018), Abstract;
Tumor starvation induced spatiotemporal control over chemotherapy for synergistic therapy: M.K. Zhang, et al.; Small 14, e1803602 (2018), Abstract;
Investigating the application of a nitroreductase-expressing transgenic zebrafish line for high-throughput toxicity testing: A.C. Chlebowski, et al.; Toxicol. Rep. 4, 202 (2017), Application(s): Use of hypoxia detection reagent with zebrafish embryos,
Modulation of alveolar macrophage innate response in proinflammatory-, pro-oxidant-, and infection- models by mint extract and chemical constituents: Role of MAPKs: N. Yadav & H. Chandra; Immunobiology 223, 49 (2017), Abstract;
Nitroimidazole derivatives of polypyridyl ruthenium complexes: Towards understanding their anticancer activity and mode of action: O. Mazuryk, et al.; Eur. J. Pharm. Sci. 101, 43 (2017), Application(s): Flow cytometry analysis using HaCaT and PANC-1 cells, Abstract;
Prodrug-embedded angiogenic vessel-targeting nanoparticle: A positive feedback amplifier in hypoxia-induced chemo-photo therapy: D. Guo, et al.; Biomaterials 144, 188 (2017), Application(s):Condocal Microscopy, Abstract; Full Text
Tumor-penetrating nanoparticles for enhanced anticancer activity of combined photodynamic and hypoxia-activated therapy: Y. Wang, et al.; ACS Nano 11, 2227 (2017), Application(s): Flow cytometry analysis of mouse breast carcinoma cells, Abstract; Full Text
Analysis of a nitroreductase-based hypoxia sensor in primary neuronal cultures: B.N. Lizama-Manibusan, et al.; ACS Chem. Neurosci. 7, 1188 (2016), Abstract;
ERK2 and CHOP restrict the expression of the growth-arrest specific p20K lipocalin gene to G0: M.J. Erb, et al.; Mol. Cell. Biol. 36, 2890 (2016), Application(s): Hypoxia levels in chick embryo fibroblasts (CEF), Abstract;
Phagocyte respiratory burst activates macrophage erythropoietin signalling to promote acute inflammation resolution: B. Luo, et al.; Nat. Commun. 7, 12177 (2016), Application(s): Flow cytometry analysis of hypoxia in exudate leukocytes and peritoneum, Abstract; Full Text
Selective advantage of trisomic human cells cultured in non-standard conditions: S.D. Rutledge, et al.; Sci. Rep. 6, 22828 (2016), Application(s): Fluorescence microscopy on human colorectal adenocarcinoma DLD1 cells, Abstract; Full Text
Low-level light in combination with metabolic modulators for effective therapy of injured brain: T. Dong, et al.; J. Cereb. Blood Flow Metab. 35, 1435 (2015), Application(s): Immunofluorescence Assay, Abstract; Full Text
Quantitative measurement of redox potential in hypoxic cells using SERS nanosensors: J. Jiang, et al.; Nanoscale 6, 12104 (2014), Abstract;
General Literature References
Fluorescent and luminescent probes for measurement of oxidative and nitrosative species in cells and tissues: progress, pitfalls, and prospects: P. Wardman; Free Radic. Biol. Med. 43, 995 (2007), Abstract;
Fluorescence probes used for detection of reactive oxygen species: A. Gomes, et al.; J. Biochem. Biophys. Methods 65, 45 (2005), Abstract;
Determination of mitochondrial reactive oxygen species: methodological aspects: C. Batandier, et al.; J. Cell. Mol. Med. 6, 175 (2002), Abstract;
Methods of detection of vascular reactive species: nitric oxide, superoxide, hydrogen peroxide, and peroxynitrite: M.M. Tarpey & I. Fridovich; Circ. Res. 89, 224 (2001), Abstract;
