Intracellular determination of mitochondrial superoxide production was performed using MitoSOX Red (Molecular Probes, Eugene, OR) as previously described.22 (link) MitoSOX Red is a fluorescent dye that permeates live cells to selectively target mitochondria superoxide which can be visualized using fluorescent microscopy. Briefly, the INS-1 cells grown on a 12-well plate were washed twice with Hank’s balanced salt solution (HBSS) (catalog number: 14065–056, Thermo Fisher Scientific) to remove the medium and incubated with 2 μM MitoSOX Red dye in the dark under culturing conditions. To confirm mitochondrial localization of MitoSOX Red, cells were incubated with 200 nM MitoTracker-Green (Molecular Probes, Eugene, OR) for 20 min following the removal of excess fluorescent dye with HBSS. Cells were then washed gently three times with warm PBS buffer and imaged (excitation/emission: 510/580 nm) immediately under a fluorescence microscope (TH4–100, Olympus). The fluorescence intensities from cells plated in 96-well plates were quantified using a BioTeck fluorescent plate reader and expressed as the mean ratio of MitoSOX to MitoTracker to compensate for differences in mitochondrial mass and unequal MitoSox loading.
Mitosox red
Manufactured by Thermo Fisher Scientific
Sourced in United States, China, United Kingdom, Germany, Japan, Canada, Australia, Italy, Switzerland, France, Spain
MitoSOX Red is a fluorogenic dye designed to measure superoxide in the mitochondria of live cells. It is readily oxidized by superoxide but not by other reactive oxygen species. The oxidized product is highly fluorescent, allowing for the detection and quantification of mitochondrial superoxide.
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Lab products found in correlation
Mitotracker green, by Thermo Fisher Scientific (176 mentions)
Cm h2dcfda, by Thermo Fisher Scientific (75 mentions)
Mitotracker green fm, by Thermo Fisher Scientific (71 mentions)
Hoechst 33342, by Thermo Fisher Scientific (52 mentions)
H2dcfda, by Thermo Fisher Scientific (52 mentions)
Facscalibur, by BD (47 mentions)
Dcfh da, by Merck Group (43 mentions)
Mitotracker red, by Thermo Fisher Scientific (39 mentions)
Mitotracker red cmxros, by Thermo Fisher Scientific (38 mentions)
Facscanto 2, by BD (33 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (32 mentions)
Lsm 700, by Zeiss (30 mentions)
Cellrox green, by Thermo Fisher Scientific (29 mentions)
Rotenone, by Merck Group (29 mentions)
Dcfh da, by Beyotime (29 mentions)
Mitotracker deep red, by Thermo Fisher Scientific (27 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (27 mentions)
Facscalibur flow cytometer, by BD (27 mentions)
Mitosox red, by BD (26 mentions)
Antimycin a, by Merck Group (25 mentions)
1 850 protocols using mitosox red
1
Intracellular Mitochondrial Superoxide Quantification
2
Mitochondrial Superoxide Detection Using MitoSox Red
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Mitochondrial superoxide was determined as previously described by using Mitosox Red (Life technologies)[22 (link)–24 (link)]. Neurons were incubated with 2 μM Mitosox Red and 200 nM mitotracker green (Life Technology) for 30 minutes followed by washing. The images of Mitosox Red staining were collected on a Nikon inverted confocal microscope with on stage incubator. The intensity was subsequently analyzed by using Nikon NIS Advanced Research software.
3
Quantification of Cytosolic and Mitochondrial ROS
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Chloromethyl‐2′7′‐dichlorodihydrofluorescein diacetate (CM‐H2CFDA) (Molecular probes, Life Technologies, Carlsbad, CA, USA) was used to detect the production of cytosolic reactive oxygen species (cROS). A total of 1 × 106 neutrophils in 1 mL were incubated in 24‐well plates for 30 minutes with CM‐H2CFDA in RPMI plus 2% autologous plasma with 25 ng/mL GM‐CSF at 37°C and 5% CO2. Cells were harvested, washed with PBS, and stimulated for 30 minutes with the indicated stimuli in HBSS at 37°C and 5% CO2. Immediately thereafter, fluorescence was assessed by flow cytometry on a BD FACS Canto II and analyzed with FlowJo software.
MitoSOX Red (Life Technologies) was used to detect the mitochondrial superoxide production in a plate reader assay. A total of 2 × 105 neutrophils in 200 µL were seeded into black flat bottom 96‐well plates (Thermo Fisher) in HBSS medium containing 25 ng/mL GM‐CSF and preincubated for 1 hour with 5 µM MitoSOX Red at 37°C and 5% CO2 and then, different stimuli added. Induction of fluorescence by mROS was detected at 2 minutes intervals with a TECAN Infinite M1000 plate reader. The area under the curve was calculated and compared to the medium control.
MitoSOX Red (Life Technologies) was used to detect the mitochondrial superoxide production in a plate reader assay. A total of 2 × 105 neutrophils in 200 µL were seeded into black flat bottom 96‐well plates (Thermo Fisher) in HBSS medium containing 25 ng/mL GM‐CSF and preincubated for 1 hour with 5 µM MitoSOX Red at 37°C and 5% CO2 and then, different stimuli added. Induction of fluorescence by mROS was detected at 2 minutes intervals with a TECAN Infinite M1000 plate reader. The area under the curve was calculated and compared to the medium control.
4
Measuring Mitochondrial ROS in Mesothelial Cells
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Mitochondrial ROS production was measured in mesothelial cells using MitoSOX Red (Life Technologies, Hillsboro, OR, USA). The dye permeates the membrane and become oxidized by superoxide exhibiting red fluorescence. Mesothelial cells from PD effluents were seeded in 48-well plates until confluence, washed with PBS and loaded with MitoSOX Red (0.8 ng/mL) in 200 µL of Hank’s Balanced Salt Solution (HBSS, Gibco, Hillsboro, OR, USA) for 30 min at 37 °C. Cells were subsequently washed, trypsinized and centrifuged and then fluorescence was measured by flow cytometry using a FACScalibur cytometer. The analysis of the data was performed using CellQuest Pro 5.1 software.
5
Measuring Mitochondrial ROS in BMDMs
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BMDM were plate in 12-well dishes, primed for 3 hr with 100 ng/mL LPS, and then stimulated in the presence or absence of pyruvate. BMDM were labeled for the last 15min of treatment with 2.5 μM MitoSOX Red (Life Technologies), collected, centrifuged for 5min at 2000 rpm at 4°C, then resuspending in ice cold PBS with 0.5% BSA and analyzed by flow cytometry (488 nm excitation, PE channel collection for MitoSOX Red). >25,000 cells were analyzed per condition.
6
Measurement of Intracellular and Mitochondrial ROS
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Intracellular reactive oxygen species (iROS) were determined using the fluorescent dye 2′,7′-dichlorofluorescein diacetate (DCFH-DA) (Molecular Probes, Eugene, OR, USA), and mitochondrial-specific ROS were measured by MitoSOX Red (Molecular Probes). Briefly, dual-biofilm cells treated with and without 12.5 μg mL−1 of MYR after centrifugation at 13,000× g for 5 min, were treated with 10 mM H2DCFDA for 1 h, or 5 M MitoSOX Red (Molecular Probes), for 30 min at 37 °C. The fluorescent cells were measured with the FACS Verse microplate reader [15 (link)].
7
Mitochondrial ROS Levels in BMDMs
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Mitochondrial reactive oxygen species (ROS) levels were measured using MitoSox-Red (Molecular Probes, Invitrogen). BMDMs were seeded in 12-well culture plates at a density of 4 × 105 cells/well. The cells were double-stained with 5 μM MitoSox-Red and 50 nM MitoTracker Green FM (Molecular Probes) in HBSS for 30 min. Images were captured at ×200 and ×400 magnifications using a fluorescence microscope (Leica). The percentage of MitoSOX-positive cells was calculated at magnification of ×200.
8
Mitochondrial ROS and Membrane Potential
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Intracellular ROS production was evaluated by labeled with 2′,7′‐dichlorodihydrofluorescein diacetate (DCFH‐DA) (10 × 10−6m ; Thermo Scientific) for 30 min. The cells were counterstained with 2.5 × 10−6m MitoSOX Red (Molecular Probes, a highly selective fluorescent dye targeting superoxide production in mitochondria in living cells) and 150 × 10−9m Mitotracker Green (Molecular Probes) for 30 min at 37 °C to detect the mitochondrial ROS. The MMP of the cells was determined by 150 × 10−9m TMRM (Molecular Probes) and 150 nM Mitotracker Green (Molecular Probes). After staining the cells for 30 min at 37 °C, they were photographed by CLSM. The corresponding fluorescent intensity was quantified and measured by the ImageJ software. Flow cytometry was further used to assess mitochondrial functions. Cells treated with different samples were collected into Eppendorf (EP) tubes and dyed with 2.5 × 10−6m MitoSOX Red (Molecular Probes) or 50 nM TMRM (Molecular Probes) for 30 min respectively, detected by an Attune NxT flow cytometer (Thermo Scientific). Quantitative analysis was performed with FlowJo 10.6 software (Tree Star, Ashland, OR, USA).
9
Mitochondrial Superoxide Assay in Parasites
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Superoxide anion (O2-) production was assayed fluorimetrically using the mitochondrial targeted probe MitoSox Red (Molecular Probes) as described previously by Carvalho et al. (2010) [31 (link)] with some modifications. Cells (107 promastigotes/mL) were loaded with 1 μM MitoSox Red for 30 min at 27°C in HBSS (Ca/Mg) (Hank's Balanced Salt Solution with calcium and magnesium, Gibco). Parasites were washed in HBSS and then treated with allicin (15–120 μM) for 3h at 27°C. After treatment cells were washed and resuspended in HBSS (107 promastigotes/mL). Aliquots of 200 μL/well were transferred to a 96-well solid black microtiter plate (Costar, Corning) and fluorescence intensity was recorded in a FLUOstar OPTIMA microplate reader (BMG Labtech) with an excitation wavelength of 510 nm and emission of 580 nm. Untreated cultures and cultures treated with 5 μM antimycin A (Sigma) were included as controls. Three experiments were carried out in triplicate.
10
Mitochondrial Superoxide Measurement by MitoSOX
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Mitochondrial superoxide production was measured using the fluorescent probe, MitoSOX Red (Molecular Probes, Invitrogen). 50,000 VM-M3 cells were seeded on 18-mm glass coverslips in 22-mm 12-well plates for 24 hours. Culture media was then replaced and treatment applied. Coverslips were then rinsed with D-PBS and stained with 5uM MitoSOX Red in Hank’s Balanced Salt Solution (HBSS) with Ca2+/Mg2+ (Gibco, Life Technologies) for 10 minutes at 37°C. Coverslips were then inverted and mounted on glass microscope slides and MitoSOX Red fluorescence (Ex/Em: 510:580 nm) was detected with a TRITC filter and a Nikon TE2000E fluorescence microscope and a 40X objective lens. The average relative fluorescence intensity of individual cells within 10 fields of view were determined for each treatment.
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