Mito-Sox Red staining (Invitrogen, USA) was used to detect the mitochondrial superoxide anion [37 ]. Mito-Sox Red staining (Invitrogen, USA) was used to detect mitochondrial superoxide anions. Images were obtained using a confocal laser scanning microscope (Nikon, A1R, Japan).
Mitosox red staining
Manufactured by Thermo Fisher Scientific
Sourced in United States
MitoSOX red staining is a fluorogenic dye that can be used to detect superoxide in the mitochondria of live cells. It is a highly selective indicator for superoxide in the mitochondria.
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Lab products found in correlation
Ros glo h2o2 assay kit, by Promega (1 mentions)
Polarstar optima, by BMG Labtech (1 mentions)
Anti hla abc fitc, by BD (1 mentions)
Epics xl, by Beckman Coulter (1 mentions)
Modfit lt 3, by BD (1 mentions)
Cellrox deep red flow cytometry assay kit, by Thermo Fisher Scientific (1 mentions)
Trypsin edta, by Merck Group (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
Elisa assay kit, by Cusabio (1 mentions)
Ros assay kit, by Beyotime (1 mentions)
Atp assay kit, by Beyotime (1 mentions)
Hoechst staining, by Beyotime (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Dcfh da, by Abcam (1 mentions)
Dm3000, by Leica (1 mentions)
Anti neun, by Cell Signaling Technology (1 mentions)
Anti bad, by Abcam (1 mentions)
Anti tuj1, by Neuromics (1 mentions)
Tunel assay, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
13 protocols using mitosox red staining
1
Mitochondrial Superoxide Detection
2
Quantifying Cellular and Mitochondrial ROS
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To evaluate cellular ROS level, ROS-Glo H2O2 assay kit (Promega, Madison, WI, USA) was employed following the manufacturer's protocol. Detectable signal was analysed using Polarstar Optima plate reader (BMG LABTECH, Ortenberg, Germany). To analyse mitochondrial ROS, MitoSOX Red staining (Thermo Fisher) was employed following the manufacturer's protocol. Briefly, 5 μmol/L of MitoSOX Red was added into medium for 10-minute incubation at 37°C and quantitatively measured using FACSCanto II.
3
Measuring Mitochondrial ROS in T/C-28a2 Cells
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Mitochondrial ROS in T/C‐28a2 cells was determined using MitoSOX Red staining (Cat#M36008; Thermo Fisher Scientific). Briefly, the cells were cultured in the chamber slides. After the specified treatment, cells were incubated with 5 μM MitoSOX for 10 min at 37°C in the dark. The mitochondrial ROS images were visualized using a fluorescent microscope. The level of mitochondrial ROS was analyzed using Image J software (NIH). Ten regions of interest were randomly selected for each group, and an average level of mitochondrial ROS was determined based on the integrated density value of the regions normalized by the number of cells in the regions.
4
Quantifying Mitochondrial Superoxide by Flow Cytometry
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Mitochondrial superoxide levels were determined by flow cytometry and MitoSOX™ Red staining (Thermo Fisher Scientific). MitoSOX™ Red is a cell-permeable dye that specifically targets mitochondria and becomes oxidized by interacting with superoxide. The fluorescent signal is a result of the binding of the oxidized dye to nucleic acids. Control samples were treated with 0.2 nM antimycin A, an inhibitor of mitochondrial electron transport complex III, to ensure that cells were capable of producing ROS. Cells were stained with fluorescent-labeled antibodies for cell identification (cancer cells were stained with anti-HLA-ABC-FITC; BD Biosciences), and primary cells with anti-CD4-FITC (Immunotech; Beckman Coulter, Inc.) and 5 µM MitoSOX™ Red for 30 minutes, and mitochondrial superoxide levels determined by flow cytometry (Epics XL; Coulter, Miami, FL, USA).
5
Mitochondrial ROS Detection by MitoSOX
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Formation of mitochondrial reactive oxygen species (ROS) was investigated via MitoSOX red staining (Invitrogen, Karlsruhe, Germany). For detection of mitochondrial ROS production, HT-22 cells were seeded in 24-well plates with 55,000 cells/well. After treatment with erastin or glutamate, respectively, cells were stained with MitoSOX red for 30 min at 37 °C. After collecting and washing once with PBS, cells were re-suspended in PBS and red fluorescence was detected by FACS analysis. Data were collected from at least 10,000 cells.
6
Quantifying Cellular Oxidative Stress
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ROS generation was determined using a CellROX Deep Red Flow Cytometry Assay Kit (Life Technologies, Carlsbad, CA, USA). The fluorescence intensity of CellROX Deep Red reflects the ROS levels. Briefly, cells were seeded on a six-well plate. After treatment, the cells were harvested with trypsin-EDTA (Sigma). The CellROX Deep Red reagent was added to the samples at a final concentration of 5 μM and incubated for 30 min at 37 °C in the dark. The medium was then removed, and the cells were washed three times with PBS. Cells were then subjected to flow cytometry using a FACS flow cytometer equipped with Modfit LT 3.0 (BD Biosciences) (excitation/emission wavelengths: 640/665 nm). Approximately 1 × 104 cells were analyzed in each of the samples.
Mitochondrial superoxide production was monitored using MitoSOX Red staining (Invitrogen, Carlsbad, CA, USA). Before use, MitoSOX Red was dissolved in DMSO to yield a 5 mM stock and subsequently diluted to a working concentration of 5 μM in warm PBS. At 24 h after treatment, cells were treated with MitoSOX red (5 μM) for 10 min at 37 °C in the dark. The cells were washed and incubated with DAPI (nuclear stain) for 5 min. Subsequently, the cells were visualized with a fluorescence microscope (Olympus, Tokyo, Japan).
Mitochondrial superoxide production was monitored using MitoSOX Red staining (Invitrogen, Carlsbad, CA, USA). Before use, MitoSOX Red was dissolved in DMSO to yield a 5 mM stock and subsequently diluted to a working concentration of 5 μM in warm PBS. At 24 h after treatment, cells were treated with MitoSOX red (5 μM) for 10 min at 37 °C in the dark. The cells were washed and incubated with DAPI (nuclear stain) for 5 min. Subsequently, the cells were visualized with a fluorescence microscope (Olympus, Tokyo, Japan).
7
Mitochondrial ROS Measurement by MitoSOX
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Formation of mitochondrial ROS was investigated via MitoSOX red staining (Invitrogen), which shows increasing red fluorescence upon reaction with mitochondrial ROS. For analysis of PHD inhibitors, HT-22 cells were seeded in 24-well plates with 55 000 cells per well. For functional analysis of PHD1 siRNAs, cells were transfected with 22 000 cells per well and grown for 48 h before treatment. At indicated time points after onset of treatment, cells were stained with MitoSOX red for 30 min at 37 °C at a final concentration of 2.5 μM. After collecting and washing once with PBS, cells were resuspended in an appropriate amount of PBS and red fluorescence was detected by FACS analysis. MitoSOX red was excited at 488 nm and emission was recorded using a 690/50 bandpass filter. Data were collected from 10 000 cells from at least three wells per condition.
8
Quantifying Oxidative Stress in Cardiomyocytes
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MitoSOX Red staining (M36008, Invitrogen, USA) was used to assess mitochondrial ROS levels in cardiac tissues. Mitochondrial ROS was detected in H9C2 cells using a ROS assay kit (S0033, Beyotime, China), and then detected using flow cytometry according to the protocol provided by the manufacturer. ATP concentration in the cardiomyocytes was measured using an ATP assay kit (S0026, Beyotime, China), following the manufacturer's instructions. The results of each assay were normalized by protein concentration as described previously [28 (link)]. The results were shown as mmol/mg protein. The levels and activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), Catalase (CAT) and malondialdehyde (MDA) were measured according to the ELISA kit instructions to further assess the level of oxidative stress (2 R-KMLJr30259, 2M-KMLJM220779 m, 2M-KMLJM220658 m, 2M-KMLJM219464 m, Camilo, China). The levels of 4-hydroxy-2-nonenal (4-HNE), 3-nitrotyrosine (3-NT), and protein carbonyl in myocardial tissues were determined using an ELISA assay kit (
CSB-E13411-13 m, Cusabio, China), and were expressed in nmol/mg protein as measured according to the manufacturer's protocol.
CSB-E13411-13 m, Cusabio, China), and were expressed in nmol/mg protein as measured according to the manufacturer's protocol.
9
Mitochondrial ROS Quantification by MitoSOX
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Mitochondrial ROS production was measured by MitoSOX Red staining (Invitrogen, Carlsbad, CA, USA, Cat# M36008). Briefly, the cells were treated with MitoSOX (5 μM), and nuclei were detected by Hoechst staining (Beyotime, Cat# C1025). Images were acquired with a Nikon 80i microscope.
10
Mitochondrial ROS Detection by Flow Cytometry
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