CellROX green (Invitrogen, Grand Island, NY, USA) was utilized for measuring oxidative stress in BAL cells. BAL cytospins were incubated with CellROX green (5 μM) for 30 min at 37 °C, fixed using 4% buffered paraformaldehyde and then mounted with ProLong Antifade with DAPI (Invitrogen, Grand Island, NY, USA). Images were obtained using confocal microscopy (Zeiss LSM 700).
Cellrox green
Manufactured by Thermo Fisher Scientific
Sourced in United States, Belgium, China, United Kingdom
CellROX Green is a fluorogenic probe that measures oxidative stress in live cells. It exhibits a green fluorescence signal upon oxidation by reactive oxygen species.
Automatically generated - may contain errors
Lab products found in correlation
Mitosox red, by Thermo Fisher Scientific (29 mentions)
Cellrox deep red, by Thermo Fisher Scientific (12 mentions)
Hoechst 33342, by Thermo Fisher Scientific (10 mentions)
Mitosox, by Thermo Fisher Scientific (7 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (7 mentions)
Cellrox orange, by Thermo Fisher Scientific (7 mentions)
Lsm 510, by Zeiss (7 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (6 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (6 mentions)
Axioscope, by Zeiss (5 mentions)
Cleaved caspase 3, by Cell Signaling Technology (5 mentions)
Lsm 700, by Zeiss (5 mentions)
Propidium iodide, by Thermo Fisher Scientific (5 mentions)
Mitotracker red, by Thermo Fisher Scientific (5 mentions)
Mitotracker green, by Thermo Fisher Scientific (5 mentions)
Caspase 3, by Cell Signaling Technology (4 mentions)
N acetylcysteine, by Merck Group (4 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (4 mentions)
Cm h2dcfda, by Thermo Fisher Scientific (4 mentions)
H2dcfda, by Thermo Fisher Scientific (4 mentions)
214 protocols using cellrox green
1
Quantifying Oxidative Stress in BAL Cells
2
Quantifying Oxidative Stress in Live Cells
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Oxidative stress in live cultures was assessed with CellROX Green (Life Technologies, #C10444), which remains non‐fluorescent until oxidized by intracellular ROS. The fluorescent signal intensity is proportional to the levels of intracellular free radicals. Cultures were plated in black optical bottom plates (Thermo Fisher, #NUN165305) and incubated in CellROX Green for 30 min after treatment, followed by 2× PBS washes prior to imaging. Images were captured on an Incucyte Zoom in phase contrast and green at 20× magnification (0.61 μm/pixel resolution) with three images per well. Mean Green Intensity was normalized to culture confluence for each image.
3
Quantifying Oxidative Stress in ARPE19 Cells
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ARPE19 cells cultured on coverslips or 96-F fluorescence microplates (black) with/without oxPOS, under control and zinc depleted and zinc-supplemented conditions, were treated with 5 μM per well CellROX green (Life Technologies) for 30 min at 37°C.
The fluorescence intensity of CellROX green in cells was quantified using a fluorescent plate reader with an excitation filter of 485 nm and an emission filter of 520 nm. The background fluorescence intensity/autofluorescence was subtracted from the CellROX green intensity of cells for data analysis. For confocal microscopy, cells were further stained with MitoTracker Red (Life Technologies) and Hoechst 33342 (Thermo Fisher Scientific Loughborough, UK) for a further 30 min.
The fluorescence intensity of CellROX green in cells was quantified using a fluorescent plate reader with an excitation filter of 485 nm and an emission filter of 520 nm. The background fluorescence intensity/autofluorescence was subtracted from the CellROX green intensity of cells for data analysis. For confocal microscopy, cells were further stained with MitoTracker Red (Life Technologies) and Hoechst 33342 (Thermo Fisher Scientific Loughborough, UK) for a further 30 min.
4
Oxidative Stress Assessment in Cell Lines
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Cells were plated in 2-well chamber slides. Cells were treated with appropriate drug concentrations for 48 hours. CellROX Green (Thermo Fisher Scientific) was added to a final concentration of 5 μmol/L. Cells were incubated in CellROX Green for 1 hour at 37°C. Cells were then washed with PBS and fixed in 3.7% formaldehyde for 15 minutes at room temperature. DAPI was used as a nuclear counterstain. Images were collected on a Zeiss Axio Vert.A1 microscope. ImageJ software was used to quantify CellROX Green mean signal intensity.
5
Measuring Cellular Oxidative Stress
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Respectively 2.5.105 and 5.105 OCI-M1 and OCI-M2 cells per well were seeded in a 6-well plate in 2 ml medium with or without 5 μM AZD1208. Following 48 hours of culture, positive and negative control cells were treated according to the manufacturer's instructions with 400 μM tert-butyl hydroperoxide (TBHP) and 5 mM N-acetyl cysteine (NAC). All samples were incubated for 1 hour with 500 nM CellROX green (ROS indicator, Molecular Probes) or 5 μM MitoSOX red (mitochondrial superoxide indicator, Molecular Probes) and subsequently analyzed by flow cytometry using 488 nm excitation for CellROX green and 510 nm excitation for MitoSOX red.
6
Cellular ROS and Mitochondrial Membrane Evaluation
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Analysis of cellular reactive oxygen species and mitochondrial membrane permeability were performed as previously described [ManshianBiomat, ManshianToxSci] . BEAS-2B and HFF-1 cells seeded in dark 96-well plates were treated with the QDs at the above listed concentrations and incubated for 24 h. Following the exposure period cells were washed and stained with CellROX Green (Molecular Probes Europe, BV, Belgium). For the InCell based analysis cells were seeded in 24-well plates and stained with CellROX Green and MitoTracker Red CMXRos (Molecular Probes Europe, BV, Belgium). Fluorescence intensity was determined using an Omega plate reader (BMG Labtech, UK) while InCell image acquisition took place on an InCell analyzer 2000 (GE Healthcare, Belgium) (for more details see supplementary information).
7
Apoptosis Pathway Activation Protocol
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Caspase-9, caspase-8, caspase-3, cleaved-caspase-3, poly(ADP-ribose) polymerase (PARP), XIAP, cIAP-1, cIAP2, Bcl-2, Bcl-xL, and Bax were procured from Cell Signaling Technologies (Beverly, MA, USA) and the GAPDH antibody was procured from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, United States). Annexin V-FITC, propidium iodide staining solution, Hoechst33342Solution, BD Cytofix/Cytoperm Plus fixation and permeabilization solution kit(BD (Pharmingen San Jose, CA, USA). The Cell Counting Kit-8 (CCK-8) kit and N-acetyl cysteine (NAC) was obtained from Sigma-Aldrich (St. Louis, MO, United States). z-VAD-FMK was bought from Calbiochem (San Diego, CA, USA). CellROXGreen, MitoSOXRed, andThiolTracker Violet were purchased from Invitrogen (Waltham, MA, USA). Mitopotential kit was purchased from the EMD Millipore Corporation (Danvers, MA, USA).
8
Evaluating Apoptosis and Oxidative Stress in hDPCs
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Apoptosis of hDPCs was evaluated using an Annexin V-FITC/propidium iodide (PI) double staining assay (BD, Bergen, NJ, USA). After treatments with melatonin and (or) H2O2, hDPCs were collected, rinsed, and incubated with Annexin V-FITC and PI for 15 min at room temperature. Cell samples were then analyzed by flow cytometry (Beckman Coulter, Brea, CA, USA). The proportions of Annexin V-positive cells were recorded as apoptotic rates.
Intracellular ROS levels were measured using CellROX® Green (Invitrogen, Grand Island, NY, USA). Upon H2O2 and melatonin treatment, hDPCs were collected and incubated with CellROX® reagent for 1 h at 37 °C, washed with PBS for three times, and analyzed by flow cytometry. Intracellular ROS levels were indicated by the mean intensity of green fluorescence.
A Mitochondrial Membrane Potential Detection Kit (JC-1; Beyotime, Shanghai, China) was employed to analyze mitochondrial membrane potential (ΔΨm) in hDPCs. After various treatments, hDPCs were collected and incubated with JC-1 staining solution for 20 min at 37 °C. After rinsing, the cells were resuspended for flow cytometry. The ratio of red (J-aggregates)/green (JC-1 monomer) fluorescence was calculated, which represents ΔΨm.
A total of 10,000 cells were harvested from each sample for these flow cytometric analyses. Experiments were repeated at least three times.
Intracellular ROS levels were measured using CellROX® Green (Invitrogen, Grand Island, NY, USA). Upon H2O2 and melatonin treatment, hDPCs were collected and incubated with CellROX® reagent for 1 h at 37 °C, washed with PBS for three times, and analyzed by flow cytometry. Intracellular ROS levels were indicated by the mean intensity of green fluorescence.
A Mitochondrial Membrane Potential Detection Kit (JC-1; Beyotime, Shanghai, China) was employed to analyze mitochondrial membrane potential (ΔΨm) in hDPCs. After various treatments, hDPCs were collected and incubated with JC-1 staining solution for 20 min at 37 °C. After rinsing, the cells were resuspended for flow cytometry. The ratio of red (J-aggregates)/green (JC-1 monomer) fluorescence was calculated, which represents ΔΨm.
A total of 10,000 cells were harvested from each sample for these flow cytometric analyses. Experiments were repeated at least three times.
9
Curcumin-induced Apoptosis in Cancer Cells
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Curcumin, CCK-8 kit, DMSO, and N-acetyl cysteine were purchased from Sigma Chemical Co (St. Louis, MO, United States) (Caspase-9, caspase-3,cleaved caspase-3,PARP,XIAP,p-Akt,Akt,GSK3,P-GSK3,FOXO1,P-FOXO1,GAPDH,cIap1,cIap2, Bcl-2, Bcl-xl, caspase 8, and cleaved caspase-8 antibodies were obtained from Cell Signaling Technologies (Beverly, MA, United States). Bax, p-H2AX, and cytochrome c antibodies and cisplatin were procured from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, United States). Annexin V fluorescein isothiocyanate (FITC), Propidium Iodide (PI), and p-H2AX (pS139) antibodies were purchased from BD Biosciences (San Jose, CA). z-VAD-FMK was obtained from Calbiochem (San Diego, CA, United States). CellROX Green was obtained from Invitrogen (MA, United States). Curcumin was dissolved in DMSO and further diluted in the cell culture media for the treatment of cells, so that the final concentration of DMSO in wells is 0.1% at the highest concentration of Curcumin used in the study. Viability assays showed that 0.1% DMSO is non-toxic to the cells (data not shown).
10
Intracellular ROS Measurement in CNT/F Exposure
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Intracellular ROS formation after 24 h post exposure of the CNT/F was assessed using CellROX® Green (Invitrogen). Cells were seeded at 46,900 cells/cm2 overnight in a 24-well plate and dosed at a concentration of 0.024, 0.24, 2.4, or 24 μg/ml of one of the nine materials tested. In terms of surface area this corresponds to 0.012, 0.12, 1.2 and 12 μg/cm2. After 24 h of exposure to CNT/F, cells were detached using Trypsin-EDTA, and washed and incubated with 50 μM CellROX for 20 min. Cells were washed and fixed by incubating them with 10% formaldehyde in PBS. The change in CellROX fluorescence was captured using a BD LSR II flow cytometer (BD Biosciences, San Diego, CA). The cells were strained through a Flowmi™ Cell Strainer (Bel-Art Products, Inc. Wayne, NJ) to achieve uniform single cell suspensions and remove any aggregates. The mean fluorescence was determined using FlowJo (FlowJo LLC, Ashland, OR). The experiment was performed on four separate days with each dose tested in triplicates each day. At least 10,000 cells were analyzed per sample in each group.
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