Cellrox deep red reagent

Manufactured by Thermo Fisher Scientific

Sourced in United States, Japan

CellROX Deep Red Reagent is a fluorogenic probe used to measure oxidative stress in live cells. It is cell-permeant and upon oxidation by reactive oxygen species (ROS), it exhibits a red fluorescent signal.

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355 protocols using cellrox deep red reagent

Oxidative Stress Measurement in Cells

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To analyze oxidative stress, treated cells were incubated with 1 µmol/L CellROX Deep Red Reagent (Thermo Fisher Scientific) at 37°C for 30 min, then stained with Hoescht (Thermo Fisher Scientific) for 15 min. Cells were washed and imaged in RPMI 1640 medium lacking phenol red. Islets were incubated with 5 μmol/L CellROX Deep Red Reagent (Thermo Fisher Scientific) at 37°C for 30 min, then stained with Hoescht (Thermo Fisher Scientific) for 15 min and fixed with 3.7% paraformaldehyde for 20 min at room temperature. Samples were imaged on a Zeiss LSM 700 laser scanning confocal microscope.

Marasco M.R., Conteh A.M., Reissaus C.A., Cupit JE V., Appleman E.M., Mirmira R.G, & Linnemann A.K. (2018). Interleukin-6 Reduces β-Cell Oxidative Stress by Linking Autophagy With the Antioxidant Response. Diabetes, 67(8), 1576-1588.

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Quantifying Oxidative Stress in Islets and Cells

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For mouse islets, following stress treatment, 50–100 intact islets were transferred to a polysterene round bottom tube and CellROX Deep Red Reagent (Invitrogen) was added at 1:500 dilution to culture media followed by incubation at 37 °C for 45 min. Islets were washed 2X with PBS, dispersed to single cell suspension, attached to slides using cytospin, and imaged using fluorescence microscopy (Keyence BZ-X700 microscope). GFP signal was used to identify transduced cells and the CellROX signal from each cell was quantified and normalized by cell area using BZ-X Advanced Analysis Software. Signal from at least 60 GFP positive cells was assessed for each condition per experiment.
For Min6 cells, following stress treatment, CellROX Deep Red Reagent (Invitrogen) was added at 1:500 dilution directly to culture media followed by incubation at 37 °C for 30 min. Cells were washed 2X with PBS and imaged using fluorescence microscopy (Keyence BZ-X700 microscope). Bright field images were used to determine cell areas and CellROX signal was quantified from at least 6 imaging fields for each group using BZ-X Advanced Analysis Software.

Good A.L., Cannon C.E., Haemmerle M.W., Yang J., Stanescu D.E., Doliba N.M., Birnbaum M.J, & Stoffers D.A. (2019). JUND regulates pancreatic β cell survival during metabolic stress. Molecular Metabolism, 25, 95-106.

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Measuring ROS in TBP/Q79-GFP cells

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293 TBP/Q₇₉-GFP cells were plated on 6-well (5 × 10⁴/well) dishes, treated with the SG-Tang (100 μg/ml) for 8 h, and induced TBP/Q₇₉-GFP expression for 6 days. Fluorogenic CellROX deep red reagent with final concentration of 5 μM (Molecular Probes, Eugene, OR, USA) was added to the cells and incubated at 37°C for 30 min. Then the cells were washed with PBS and analyzed by a flow cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA) with excitation/emission wavelengths at 488/507 nm (green, TBP/Q₇₉-GFP expression) and 640/665 nm (red, ROS). For each sample, 5 × 10⁴ cells were analyzed.

Chen C.M., Chen W.L., Hung C.T., Lin T.H., Lee M.C., Chen I.C., Lin C.H., Chao C.Y., Wu Y.R., Chang K.H., Hsieh-Li H.M, & Lee-Chen G.J. (2019). Shaoyao Gancao Tang (SG-Tang), a formulated Chinese medicine, reduces aggregation and exerts neuroprotection in spinocerebellar ataxia type 17 (SCA17) cell and mouse models. Aging (Albany NY), 11(3), 986-1007.

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Oxidative Stress Imaging in Islet Cells

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Islets were plated onto CellCarrier-96 ultra microplates (Perkin Elmer) in phenol red-free RPMI 1640 medium supplemented with 10% FBS, 1% penicillin/streptomycin, 100 µg/ml primocin, 10 mM Hepes, and 1 mM sodium pyruvate one day prior to staining. Islets were treated with menadione (AdipoGen Life Science, 10 µM) for 3 hr at 37°C. Islets were stained with CellROX Deep Red reagent (Molecular Probes, C10422, 100 µM) and Hoechst 33342 (Invitrogen, 10 µg/ml) for 1 hr at the same time. Islets were washed three times with HBSS and incubated in HBSS for imaging while temperature and CO₂ were controlled. Images were obtained by an Opera Phenix high content screening system (63X objective lens) in the SBP High Content Screening (HCS) Facility and seven z-stack images (1 µm interval) were combined.

Jang I., Pottekat A., Poothong J., Yong J., Lagunas-Acosta J., Charbono A., Chen Z., Scheuner D.L., Liu M., Itkin-Ansari P., Arvan P, & Kaufman R.J. (2019). PDIA1/P4HB is required for efficient proinsulin maturation and ß cell health in response to diet induced obesity. eLife, 8, e44528.

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Measuring Cellular and Lipid ROS

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General oxidative stress indicator CM-H2-DCFDA (0.1 mmol/L, Molecular Probes) or CellROX Deep Red Reagent (1:500, Molecular Probes) was used to measure cellular ROS levels. Lipid ROS was detected by staining cells with lipid peroxidation sensor BODIPY 581/591 C11 (2 μmol/L, Molecular Probes). Briefly, cells grown in 2-D adherent conditions or 3-D cultures (48–72 hours for Ewing sarcoma cells) were dissociated with CellStripper (A673 cells) or 0.25% trypsin-EDTA (all other cell lines), and the cells were resuspended in normal growth media containing those reagents or DMSO (as a negative control). The samples were incubated for 30 minutes (lipid ROS) or 45 minutes (general ROS) at 37°C. Then, cells were washed, resuspended in cold PBS, and analyzed on a BD FACSCalibur flow cytometer by FL-1 channel (CM-H2-DCFDA and BODIPY 581/591 C11) or FL-4 channel (CellROX Deep Red). Data were analyzed using FlowJo v10 (FlowJo LLC), and the percentage of lipid ROS-high cells (arbitrary based on the intensity of controls) and median fluorescence intensity of general ROS were assessed.

Zhang H.F., Hughes C.S., Li W., He J.Z., Surdez D., El-Naggar A.M., Cheng H., Prudova A., Delaidelli A., Negri G.L., Li X., Ørum-Madsen M.S., Lizardo M.M., Oo H.Z., Colborne S., Shyp T., Scopim-Ribeiro R., Hammond C.A., Dhez A.C., Langman S., Lim J.K., Kung S.H., Li A., Steino A., Daugaard M., Parker S.J., Geltink R.I., Orentas R.J., Xu L.Y., Morin G.B., Delattre O., Dimitrov D.S, & Sorensen P.H. (2021). Proteomic Screens for Suppressors of Anoikis Identify IL1RAP as a Promising Surface Target in Ewing Sarcoma. Cancer Discovery, 11(11), 2884-2903.

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Measuring Microglia Oxidative Stress

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ROS generation was measured by labeling cells with CellROX Deep Red Reagent following the manufacturer’s instructions (Molecular Probes, Life Technologies). CellROX deep red is a cell permeable non-fluorescent dye that is oxidized by cytoplasmic free radicals to emit fluorescence. Microglia were grown on 35-mm glass bottom dishes (MatTeK Corps). After various treatments, cells were washed twice and loaded with 5 μM of the fluorescent probe for 1 h at 37 °C. Cells were washed three times with PBS and imaged immediately. Multiple random images were captured using an Olympus FV1000 confocal microscope equipped with a HeNe 635 nm laser, 20× objective, NA 0.75 with an electronic zoom of 1.2. Laser intensity, scan speed, and other settings were attenuated to minimize phototoxicity and photobleaching. In addition, for reproducibility and comparison purposes, all microscope settings were kept identical across all treatment groups.

Akhtar F., Rouse C.A., Catano G., Montalvo M., Ullevig S.L., Asmis R., Kharbanda K, & Maffi S.K. (2017). Acute maternal oxidant exposure causes susceptibility of the fetal brain to inflammation and oxidative stress. Journal of Neuroinflammation, 14, 195.

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Measuring Intracellular ROS Levels

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ROS levels were measured using CellRox Deep Red Reagent (Molecular Probes) as an indicator of intracellular ROS. For microscopic analysis, GFP labeled SKOV3ip1 cells were cultured with CAFs or alone on poly-D-lysine coated cover glass for 4 hr. Cells were then loaded with 5 μM CellRox Deep Red for 30 min, washed with PBS and imaged live using a Zeiss 510 confocal microscope. Quantification from 3 independent experiments is shown. Representative images are shown.

Curtis M., Kenny H.A., Ashcroft B., Mukherjee A., Johnson A., Zhang Y., Helou Y., Batlle R., Liu X., Gutierrez N., Gao X., Yamada S.D., Lastra R., Montag A., Ahsan N., Locasale J.W., Salomon A.R., Nebreda A.R, & Lengyel E. (2018). Fibroblasts mobilize tumor cell glycogen to promote proliferation and metastasis. Cell metabolism, 29(1), 141-155.e9.

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Oxidative Stress Assay in Macrophages

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hMacs and iMacs, cultured in black 96-well clear-bottom plates or glass chamber slides, were stained with CellROX Deep Red Reagent (Molecular Probes) at a final concentration of 5 μM. After feeding macrophages with IgG-opsonized Gaucher erythrocyte ghosts in the presence or absence of diphenyleneiodonium (Sigma) at a final concentration of 5 μM, they were incubated for 30 min at 37°C. Cells were washed, and fluorescence was evaluated at 640/665 nm using a FlexStation Multi-Mode microplate reader. Cells, cultured on glass coverslip chamber slides, were stained with CellROX, and live-cell imaging was performed using Zeiss 510 META laser scanning microscope (×63).

Aflaki E., Stubblefield B.K., Maniwang E., Lopez G., Moaven N., Goldin E., Marugan J., Patnaik S., Dutra A., Southall N., Zheng W., Tayebi N, & Sidransky E. (2014). Macrophage Models of Gaucher Disease for Evaluating Disease Pathogenesis and Candidate Drugs. Science translational medicine, 6(240), 240ra73.

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Reagents and Materials for Cell Studies

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Sodium dichromate dehydrate (Na₂Cr₂O₇) was from Sigma (St Louis, MO). Cisplatin was from Enzo Life Sciences (Farmingdale, NY). Dulbecco’s modified Eagle’s medium (DMEM), Defined keratinocyte serum-free medium, fetal bovine serum (FBS), and Oligo (dT)₂₀ and AccuPrime Taq DNA Polymerase High Fidelity were from Invitrogen (Carlsbad, CA). Bradford Protein Assay Reagent was from Bio-Rad (Hercules, CA). RNeasy Mini kit and plasmid prep kit were from Qiagen (Valencia, CA). M-MLV reverse transcriptase was from Promega (Madison, WI). Perfecta Sybr Green Fastmix was from Quanta Biosciences (Gaithersburgh, MD). 5-(and -6)-chloromethyl-2, 7-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H₂DCFDA) and CellROX deep red reagent were from Molecular Probes (Eugene, OR). Antibodies against NQO1, GAPDH, and β-actin were obtained from Santa Cruz (Santa Cruz, CA). Antibody against FBP1 was from Sigma (St Louis, MO). Antibody against SOD2 was from Millipore (Billerica, MA). Antibodies against Notch1, p21, cyclin D1, Bcl-2, Bcl-xL, PARP, AP1/c-jun, cleaved caspase 3, and cleaved caspase 9 were from Cell Signaling Tech (Danvers, MA). Enhanced chemiluminescence reagent was from Amersham (Pittsburgh, PA). Matrigel was from BD Biosciences (San Jose, CA).

Dai J., Ji Y., Wang W., Kim D., Fai L.Y., Wang L., Luo J, & Zhang Z. (2017). Loss of fructose-1,6-bisphosphatase induces glycolysis and promotes apoptosis resistance of cancer stem-like cells: an important role in hexavalent chromium-induced carcinogenesis. Toxicology and applied pharmacology, 331, 164-173.

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Quantifying Oxidative Stress Levels in Spermatocytes

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Oxidative stress was detected by using CellROX deep red reagent and MitoSOX red mitochondrial superoxide indicator (both from Molecular Probes, Eugene, MN, USA). CellROX exhibits fluorescence (emission maxima at 650 nm) upon oxidation by reactive oxygen species, and MitoSOX red (emission maxima at 580 nm) detects superoxide in the mitochondria of live cells. Spermatocytes were treated with CSC or DMSO for 1 h and 5 h as previously reported.^{13 (link)} Then, CellROX and MitoSOX reagents were added to a final concentration of 1 μM and 5 μM, for 30 and 10 min, respectively, at 37 °C. The cells were washed with warm buffer as per the manufacturer’s protocol, counter-stained with SYBR 14 dye or TO-PRO-3 (Molecular Probes; 1 : 1,000) for 5 min at room temperature, and then subjected to flow cytometry.

Esakky P., Hansen D.A., Drury A.M., Cusumano A, & Moley K.H. (2015). Cigarette smoke-induced cell death of a spermatocyte cell line can be prevented by inactivating the Aryl hydrocarbon receptor. Cell Death Discovery, 1, 15050-.

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