Cellrox orange

Manufactured by Thermo Fisher Scientific

Sourced in United States, Japan

CellROX Orange is a fluorogenic probe used to detect oxidative stress in live cells. It exhibits a bright orange fluorescence upon oxidation by reactive oxygen species (ROS) within the cell.

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Lab products found in correlation

Cellrox green, by Thermo Fisher Scientific (7 mentions) Mitosox red, by Thermo Fisher Scientific (5 mentions) Mitotracker green, by Thermo Fisher Scientific (5 mentions) Cellrox deep red, by Thermo Fisher Scientific (4 mentions) Accuri c6 flow cytometer, by BD (4 mentions) Trypsin edta, by Thermo Fisher Scientific (3 mentions) Axioskop 40, by Zeiss (3 mentions) Hoechst 33342, by Thermo Fisher Scientific (3 mentions) Sytox green, by Thermo Fisher Scientific (2 mentions) Cellrox deep red, by BD (2 mentions) Cellrox orange, by BD (2 mentions) Aria 3, by BD (2 mentions) Fluorescence microscopy, by Keyence (2 mentions) Cellrox orange, by Keyence (2 mentions) Lsrfortessa x 20 flow cytometer, by BD (2 mentions) Lsm 700 confocal microscope, by Zeiss (2 mentions) Draq5, by Cell Signaling Technology (2 mentions) Fv10i confocal microscope, by Olympus (2 mentions) Zoe fluorescent cell imager, by Bio-Rad (2 mentions) Mowiol, by Merck Group (2 mentions)

Close spelling variants of this product

CellROX (153 citations) CellROX Orange Reagent (64 citations) CellROX Orange Oxidative Stress Reagent (2 citations) CellROX® Orange ROS Detection Assay (2 citations) CellRox Orange dye (2 citations) CellROX® Orange or Green Reagent (2 citations)

49 protocols using cellrox orange

Cellular ROS Quantification by Flow Cytometry

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Cellular ROS levels were estimated by flow cytometry (BD FACSCalibur, BD Biosciences) after incubation of cells with the cell-permeable ROS indicator CellROX Orange (Invitrogen C10443). Unstimulated cells grown under standard culture conditions were washed with PBS and harvested with enzyme-free cell-dissociation buffer. Cells were pelleted at 300 g for 3 min and resuspended in Hanks’ balanced salt solution (Invitrogen 14025) containing 10 mM HEPES (Invitrogen 15630) and 5μM CellROX Orange for 30 min in an incubator. Cells were washed with Hanks’ balanced salt solution containing 10 mM HEPES, and the average fluorescence intensity of 20,000 cells was measured for each cell line.

Orr A.L., Kim C., Jimenez-Morales D., Newton B.W., Johnson J.R., Krogan N.J., Swaney D.L, & Mahley R.W. (2019). Neuronal Apolipoprotein E4 Expression Results in Proteome-Wide Alterations and Compromises Bioenergetic Capacity by Disrupting Mitochondrial Function. Journal of Alzheimer's Disease, 68(3), 991-1011.

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Zebrafish Developmental Assays

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The following reagents were used at indicated working concentrations: Acetone (50% in Ethanol, Sigma-Aldrich, #179124), CellROX Orange (2.5 μM in E3, Invitrogen, #C10443), Dimethyl sulfoxide (0.2% in E3, Sigma-Aldrich, #76855), Ethanol (50% in Acetone, Sigma-Aldrich, #459828), TMR Red in situ cell death detection kit (Sigma-Aldrich, #12156792910), low melting-point agarose (2% in E3, Gold biotechnology, #A-204-100), methanol (varying concentrations, Fisher Chemical, #A452-4), Metronidazole (5 mM in 0.2% DMSO/E3, Acros Organics, #10691063), Paraformaldehyde (4% in PBS, Sigma-Aldrich, #441244), Phosphate-buffered saline (Sigma-Aldrich, #79382), Proteinase K (10 μg/mL in PBS-T, Roche Diagnostics, #10954400), SYTOX Green (0.2 μM in E3, Invitrogen, #S7020), Tricaine (0.2 mg/mL in E3, Sigma-Aldrich, #E10521), Trypsin/EDTA (0.25% in PBS, MSKCC media core), Tween-20 (0.1% in PBS, Sigma-Aldrich, #P1379), TURBO-DNAse (4–10 U/mL in 50 mM Tris-HCL, pH 7.5, 1 mg/mL BSA, Thermo Fisher, #AM1907).

Stoddard M., Huang C., Enyedi B, & Niethammer P. (2019). Live imaging of leukocyte recruitment in a zebrafish model of chemical liver injury. Scientific Reports, 9, 28.

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Quantifying Intracellular ROS in HUVECs

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To detect intracellular ROS, HUVECs stimulated with VEGF were incubated with 10 μM CM–H₂DCFDA [5-(and 6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester, Invitrogen] for 6 min at 37 °C, fixed with 4% paraformaldehyde for 10 min at room temperature, and then mounted with VECTASHIELD Mounting Medium with DAPI. DCF fluorescence was measured by confocal microscopy (Zeiss) using the identical setting and the same exposure condition in each experiment. Relative DCF fluorescence with DAPI positive cells were analyzed using Image J (NIH). We confirmed that DCF fluorescence was abolished by incubation with adeno-catalase suggesting that DCF signal mainly detects intracellular H₂O₂, as we reported [30 (link)]. In some experiments to detect cellular redox status, we also used cell-permeant fluorescence indicator, CellROX Orange (Invitrogen). Cells were incubated with 5 μM CellROX for 30 min and fluorescence images were taken using confocal microscopy, as previously described with minor modifications [32 (link)].

Nagarkoti S., Kim Y.M., Ash D., Das A., Vitriol E., Read T.A., Youn S.W., Sudhahar V., McMenamin M., Hou Y., Boatwright H., Caldwell R., Essex D.W., Cho J., Fukai T, & Ushio-Fukai M. (2022). Protein disulfide isomerase A1 as a novel redox sensor in VEGFR2 signaling and angiogenesis. Angiogenesis, 26(1), 77-96.

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Fluorescent Labeling of Larval Zebrafish

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TG(fabp10:NTR-mTq2) larvae were reared and treated with 5 mM Mtz as described above. Immediately before imaging, larvae were placed in E3 medium containing 0.2% DMSO and the specified fluorescent dye (Table 1). After dye incubation, larvae were washed 3 times with 0.2 mg/mL Tricaine in E3, embedded in low-melting point agarose, and imaged.Table 1

Fluorescent Reporter Dye Concentrations and Incubation Times.

Fluorescent Dye	Final concentration (in E3)	Incubation time (min)
SYTOX Green (Invitrogen)	0.2 μM	20
CellROX Orange (Invitrogen)	2.5 μM	30

Stoddard M., Huang C., Enyedi B, & Niethammer P. (2019). Live imaging of leukocyte recruitment in a zebrafish model of chemical liver injury. Scientific Reports, 9, 28.

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Epididymal Sperm Oxidative Stress and Mitochondrial Permeability

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For ROS detection, mature spermatozoa collected from the cauda epididymis (from mice at age of 9–16 weeks) were incubated in mHTF medium for 90 min at 37 °C, with or without 0.006% H₂O₂ for the last 60 min and with 1 μM CellROX Orange (Invitrogen) for the last 30 min. Analysis of mPTP opening was performed with the use of a Mitoprobe Transition Pore Assay Kit (Molecular Probes). Mature spermatozoa collected from the cauda epididymis were incubated in mHTF medium for 2 h at 37 °C, with 0.03 μM calcein-AM, 4.8 μM CoCl₂, or 10 μM A23187 being added for the last 15 min. For removal of dead cells, spermatozoa were incubated with DAPI (6 μg/ml) for 5 min. Flow cytometric analysis was performed with a FACSVerse instrument (BD). The data were analyzed with the use of FlowJo V10 software.

Mise S., Matsumoto A., Shimada K., Hosaka T., Takahashi M., Ichihara K., Shimizu H., Shiraishi C., Saito D., Suyama M., Yasuda T., Ide T., Izumi Y., Bamba T., Kimura-Someya T., Shirouzu M., Miyata H., Ikawa M, & Nakayama K.I. (2022). Kastor and Polluks polypeptides encoded by a single gene locus cooperatively regulate VDAC and spermatogenesis. Nature Communications, 13, 1071.

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Oxidative Stress and Cell Death Analysis

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Following drug treatment, CellROX Orange or CellROX Green (Invitrogen) was added to each well at a final concentration of 5 μM according to the manufacturer’s instructions. For cell death experiments cells were resuspended in 200 μL of HBSS, with Annexin V-Alexa Fluor 488 (5 μL stock/100 μL cell suspension) and Sytox Red (5 nM final concentration) added and samples incubated for 15 min in the dark. Samples were analyzed on a BD LSR Fortessa X-20 flow cytometer. A total of 20,000 single cell events were recorded. Data were gated post-acquisition to include only single cells, and the corresponding fluorescence histograms and median fluorescence intensities (MFI) were extracted using FlowJo software (v. 10.1).

McCormick P.N., Greenwood H.E., Glaser M., Maddocks O.D., Gendron T., Sander K., Gowrishankar G., Hoehne A., Zhang T., Shuhendler A.J., Lewis D.Y., Berndt M., Koglin N., Lythgoe M.F., Gambhir S.S., Årstad E, & Witney T.H. (2018). Assessment of tumor redox status through (S)-4-(3-[18F]fluoropropyl)-l-glutamic acid positron emission tomography imaging of system xc- activity. Cancer research, 79(4), 853-863.

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Measuring ROS in Microglia and BV2 Cells

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To assess ROS generation in primary microglia, cell homogenates from 3- and 20-month-old male wild type mice and from 9-month old male GRN^−/− mice were prepared and antibody staining was performed as described above (see Microglia Isolation). Cell homogenates were incubated in FACS buffer with CellROX™ Deep Red (1:500, Invitrogen) for 30 min at 37°C, washed twice in FACS buffer, and CellROX™ Deep Red Intensity was analyzed on ARIA 3.1 (BD Biosciences).
To measure ROS in BV2 cells, cells were split into 24 well plates at 5×10⁴ cells per well in DMEM +5% FBS and treated with LPS, Triacsin C and vehicle solutions for 18 hours. Next, cells were incubated in DMEM+5%FBS with CellROX™ Orange (1:500, Invitrogen) for 30 min at 37°C, washed twice in PBS, and CellROX™ Orange Intensity was examined by fluorescence microscopy (Keyence Corp., Osaka, Japan). Following microscopic analysis, cells were detached using TripLE, transferred to FACS tubes and CellROX™ Orange intensity was analyzed on BD AccuriC6 flow cytometer.

Marschallinger J., Iram T., Zardeneta M., Lee S.E., Lehallier B., Haney M.S., Pluvinage J.V., Mathur V., Hahn O., Morgens D.W., Kim J., Tevini J., Felder T.K., Wolinski H., Bertozzi C.R., Bassik M.C., Aigner L, & Wyss-Coray T. (2020). Lipid droplet accumulating microglia represent a dysfunctional and pro-inflammatory state in the aging brain. Nature neuroscience, 23(2), 194-208.

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Mitochondrial ROS Overproduction in TM Cells

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To determine if mitochondrial alterations are associated with ROS overproduction, cultured TM cells (passages 1–3) were treated with CellROX Orange (Invitrogen) to evaluate the resulting ROS-sensitive fluorescent product. Briefly, the cells were incubated with 5 μM CellROX Orange for 30 minutes and then examined by an inverted phase contrast light and fluorescence microscope attached to a 37° C chamber. Live-cell imaging (epifluorescent microscope interfaced with imaging software LAS X; Leica) was performed using a ×20 objective at 545 or 565 nm to detect the baseline. Another group of TM cells was challenged with 200 μM hydrogen peroxide (H₂O₂) for 30 minutes. After assessment of H₂O₂ concentrations (50–300 μM), we determined that lower concentrations of H₂O₂ required extended exposure times (2–4 hours), which were poorly tolerated by the sensitive TM cells in culture with fetal bovine serum-free medium. Reactive oxygen species accumulation was measured and compared with the baseline. All experiments were performed in triplicate, and the fluorescence intensity was quantified by ImageJ version 1.51a (National Institute of Health).

Wu H., Shui Y.B., Liu Y., Liu X, & Siegfried C.J. (2021). Trabecular Meshwork Mitochondrial Function and Oxidative Stress: Clues to Racial Disparities of Glaucoma. Ophthalmology Science, 2(1), 100107.

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Lysosomal Oxidative Stress Quantification

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WT and AEP^−/− MEF grown on FluoroDish dishes (World Precision Instruments Inc.) treated as indicated with the different inhibitors were incubated with 100 nM LysoTracker Green DND26 (Invitrogen) for 2 h at 37 °C and then with 2.5 μM CellROX Orange (Invitrogen) for 30 min at 37 °C. The cells were washed twice with PBS, fresh complete DMEM was added to the dishes and live cell imaging carried out at 37 °C and 5% CO₂ using a LSM 700 confocal microscope (Carl Zeiss). CellROX Orange intensity overlapping with LysoTracker Green DND26 positive structures was quantified using the Volocity 3D Image Analysis Software (PerkinElmer). The mean intensity of CellROX Orange per LysoTracker Green DND26 positive structure, normalised for lysosomal size (µm²) and per cell was calculated and the standard error calculated using 15 cells per experimental condition.

Martínez-Fábregas J., Prescott A., van Kasteren S., Pedrioli D.L., McLean I., Moles A., Reinheckel T., Poli V, & Watts C. (2018). Lysosomal protease deficiency or substrate overload induces an oxidative-stress mediated STAT3-dependent pathway of lysosomal homeostasis. Nature Communications, 9, 5343.

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Quantifying ROS in Ovarian Cancer Cells

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ROS were detected in human ovarian cancer cell lines using the cell permeable fluorophore CellROX Orange (Invitrogen, C10443). A2780 WT, CisR and DoxR were seeded in 6-well plates at a density of 5 × 10⁵ cells per well in 2 mL media 24 h prior to performing the assay. PEO 1, PEO 4 and PEO 6 cells were seeded at densities of 2 × 10⁵, 6 × 10⁵ and 8 × 10⁵ cells per well (2 mL media), respectively, 48 h prior to assay to ensure complete cell attachment. Fresh media supplemented with 2 mM glutamine was added to each 6-well plate one hour before the experiment. A final concentration of 1 μM CellROX Orange reagent was added to each well and incubated for 30 min at 37 °C, protected from light. Cells were then washed with PBS, harvested using 0.05% trypsin-EDTA (Thermo Fisher Scientific; 25300062) and suspended in 1 mL of ice-cold Hanks balanced salt solution (ThermoFisher Scientific; 14025092). The cell suspension was passed through a 35 μM filter and kept on ice prior to analysis on a LSRFortessa™ X-20 flow cytometer (561 nm laser and 586/15 bandpass filter; BD Biosciences), with 20,000 single cell events recorded per sample. Laser power remained constant between matched cell lines but was adjusted for A2780 vs. PEO lines. Data were gated post-acquisition based on forward (FS) and side scattering (SS) profiles to include only single cell events and to exclude cellular debris.

Greenwood H.E., McCormick P., Gendron T., Glaser M., Pereira R., Maddocks O.D., Sander K., Zhang T., Koglin N., Lythgoe M.F., Årstad E., Hochhauser D, & Witney T.H. (2019). Measurement of tumor antioxidant capacity and prediction of chemotherapy resistance in preclinical models of ovarian cancer by positron emission tomography. Clinical cancer research : an official journal of the American Association for Cancer Research, 25(8), 2471-2482.

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