Cyto-ID Green dye and MitoSox were purchased from Enzo Life Sciences, Inc. (Farmingdale, NY, USA). LysoTracker was purchased from Invitrogen (SanDiego, CA, USA). FITC-AnnexinV/PI kit was purchased from BD Biosciences (Franklin Lakes, NJ, USA). The mouse antibody to ASS was obtained from BD Biosciences. All the other primary antibodies including antibodies to β-actin, LC3B, cleaved caspase 3, PARP, anti-cleaved PARP, phospho-mTOR (Ser2448), phospho-Akt (Ser473), p70S6 kinase phospho (pS371), phospho-4EBP1-pT45 were obtained from Cell Signaling Technology (Danvers, MA, USA). The secondary antibodies were purchased from MR Biotech (Shanghai, China). The autophagy inhibitors of the PI3K inhibitor LY294002 and the lysosomal inhibitor CQ, 3-(4,5-dimetrylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) as well as L -arginine were purchased from Sigma-Aldrich (St. Louis, MO, USA). Z-VAD-fmk and NAC were obtained from Beyotime Institute of Biotechnology (Haimen, Jiangsu Province, China).
Mitosox
Manufactured by Enzo Life Sciences
Sourced in United States
MitoSOX is a fluorogenic dye designed to measure superoxide (O2•−) in the mitochondria of live cells. It is rapidly and selectively targeted to the mitochondria and oxidized by superoxide, but not by other reactive oxygen species (ROS), to yield a red fluorescent product.
Automatically generated - may contain errors
Lab products found in correlation
Mitosox, by Thermo Fisher Scientific (3 mentions)
Mitosox red, by Thermo Fisher Scientific (3 mentions)
Total ros detection kit, by Enzo Life Sciences (2 mentions)
Color matched beads, by BD (2 mentions)
Tom20 antibodies, by Novus Biologicals (2 mentions)
Cd66b clone g10f5, by BioLegend (2 mentions)
Dnase, by Roche (2 mentions)
Mitosox, by BD (2 mentions)
Leica dm6000b fluorescence microscope, by Leica Microsystems (2 mentions)
Mito tempo, by Enzo Life Sciences (2 mentions)
Cyto id green dye, by Enzo Life Sciences (1 mentions)
3 4 5 dimetrylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Merck Group (1 mentions)
Z vad fmk, by Beyotime (1 mentions)
Anti cleaved parp, by Cell Signaling Technology (1 mentions)
Lysotracker, by Thermo Fisher Scientific (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
L arginine, by Merck Group (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Ly294002, by Merck Group (1 mentions)
N acetyl cysteine (nac), by Beyotime (1 mentions)
7 protocols using mitosox
1
Autophagy and Apoptosis Regulation
2
Assessing Mitochondrial Function in Neutrophils
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The total ROS detection kit and MitoSOX (2.5 µM) were used according to the manufacturer’s instructions (Enzo Life Sciences and Life Technologies, NY). Lupus LDGs and control NDGs were incubated with either MitoSOX (5 µM) or mitoTracker® green FM (100 nM) and cells were quantified by flow cytometry following manufacturer’s instructions for concentration and timing and using BD color matched beads for compensation (catalog number: 552843 BD Biosciences, San Diego, CA). Mitochondria were also analyzed by flow cytometry using TOM20 antibodies (Novus Biologicals) and MitoSOX (Life Technologies). In some experiments, neutrophils were treated with pronase (2 mg/mL, Calbiochem, San Diego, CA) or DNase (50 µg/mL, Roche) 30 minutes prior to addition of TOM20. Neutrophil activation was assessed by cell surface expression of CD66b (clone G10F5, BioLegend). Phagocytosis of 8-OHdG ICs was analyzed by flow cytometry and immunofluorescence microscopy upon incubation of neutrophils with Alexa Fluor-conjugated 8-OHdG ICs (2.5 µg/mL, pre-formed as described above) for 30 minutes. Phagocytosis index was determined as phagocytosed ICs per neutrophil and field of observation. Data were analyzed by FlowJo (Tree Star Inc, Ashland, OR). For all analyses, isotype antibodies or fluorescently labeled beads were used as negative controls.
3
Assessing Mitochondrial Function in Neutrophils
4
Measuring Mitochondrial ROS in Cells
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To measure mitochondrial reactive oxygen species (ROS), the fluorescent probe MitoSOX Red (Life Technologies) was used as previously described.32 (link) In brief, BAR-10T cells were placed in 2-well, Lab-Tek II chamber slides (Nalge Nunc, Rochester, NY) with a chamber volume of 1 mL at 1 × 105 cells per well. Cells were treated with or without 1 μg/mL LPS for 24 hours, after which the cells were washed 2 times with phosphate-buffered saline (PBS). Cells were loaded with 5 μmol/L MitoSOX Red for 10 minutes at 37°C, and then washed 2 times with PBS. Cells were fixed in 2% paraformaldehyde for 3–5 minutes, and washed 2 times with PBS. Then the cells were stained with 4′,6-diamidino-2-phenylindole for 1 minute and washed with PBS 3 times. For some studies, cells were treated with 100 μmol/L Mito-TEMPO (Enzo Life Sciences, Farmingdale, NY) and LPS for 24 hours followed by loading with MitoSOX as described earlier. Cells were imaged with a Leica DM6000 B fluorescence microscope (Leica Microsystem, Buffalo Grove, IL) and fluorescence was quantitated using National Institutes of Health ImageJ software (version 1.48; Bethesda, MD) from 5 separate high-power fields (40×) per well, and then averaged. All assays were performed in at least 2 independent experiments.
5
Mitochondrial Oxidative Stress Assay
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SH-SY5Y control, Parkin O/E and Parkin O/E;SREBP2(−/−) cells were grown in galactose-supplemented media prior to staining with MitoSOX Red™ (Invitrogen, USA) and rotenone treatment. Cells were co-stained with 5 μM MitoSOX, Hoechst (1:1000; Enzo Life Sciences, USA) and BODIPY 493/503 (Invitrogen, USA) (1:1000) in culture media for 10 min at 37°C. Cells were rinsed and imaged in HBSS without phenol red (Gibco, USA) supplemented with Ca2+ and Mg2+. Live confocal fluorescence imaging was performed on a spinning disc confocal (SDC) setup built around a Nikon Ti2 inverted microscope equipped with a Yokogawa CSU-W1 confocal spinning head, a Plan-Apochromat objective (100×, 1.45 NA) and a back-illuminated sCMOS camera (Prime 95B; Photometrics, USA). All image acquisitions were carried out using MetaMorph (Molecular Device, USA) with exposure time 500 ms. Automated MitoSOX intensity measurements were done using MatLab algorithm (66 (link)) and is available upon request.
6
Mitochondrial ROS Measurement in Cells
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BMDMs were plated in half-area black 96-well plates in RPMI without phenol red (Invitrogen). Cells were incubated for 10 min at 37°C with 5 μM MitoSOX (Thermofisher). Stimuli were added and fluorescence (Ex510, Em580) recorded using a Synergy plate reader (BioTek, Winooski, VT). Neutrophils in HBSS without calcium and magnesium (ThermoFisher) were incubated for 1 h with 100 nM PMA (Enzo Life Sciences, Farmingdale, NY) and 5 μM MitoSOX during the last 10 min of incubation. ROS were measured by flow cytometry.
7
Mitochondrial ROS Measurement using MitoSOX
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