NGR1 (molecular weight = 933.15; purity > 98%) was purchased from Shanghai Winherb Medical S and T Development (Beijing, China). Dulbecco’s modified Eagle’s medium/F12 (DMEM/F12) and foetal bovine serum (FBS) were obtained from Gibco (Grand Island, NY, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and fluorescent dye JC-1 were acquired from Enzo Life Sciences (New York, NY, USA). ELISA kits for 4-hydroxynonenal (4-HNE), 8-hydroxy-2′-deoxyguanosine (8-OHdG) and protein carbonyl were acquired from Expandbio (Beijing, China). An Annexin V/propidium iodide (PI) kit, MitoTracker® Red CM-H2XRos and a MitoSOX™ assay kit purchased from Invitrogen (Grand Island). A terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling (TUNEL) detection kit was purchased from Roche Diagnostics GmbH, Mannheim, Germany. Cell protein extraction kits, PINK1 siRNA, and control siRNA were provided by Santa Cruz Biotechnology (Dallas, TX, USA). Bicinchoninic acid assay kits were purchased from Pierce Biotechnology (Waltham, MA, USA). The pCMV-G FP-LC3 expression vector was acquired from Cell Biolabs (San Diego, CA, USA).
Mito sox assay kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Mito-Sox Assay Kit is a fluorescent-based detection system for measuring mitochondrial superoxide levels in live cells. The kit provides the necessary reagents to stain and quantify mitochondrial superoxide production.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 800, by Zeiss (2 mentions)
Ros assay kit, by Beyotime (2 mentions)
Hoechst, by Beyotime (2 mentions)
Dulbecco s modified eagle s medium f12 dmem f12, by Thermo Fisher Scientific (1 mentions)
Pcmv gfp lc3 expression vector, by Cell Biolabs (1 mentions)
Foetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Mitotracker red cm h2xros, by Thermo Fisher Scientific (1 mentions)
Pink1 sirna, by Santa Cruz Biotechnology (1 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Enzo Life Sciences (1 mentions)
Control sirna, by Santa Cruz Biotechnology (1 mentions)
Bicinchoninic acid assay kit, by Thermo Fisher Scientific (1 mentions)
Fluorescence microscopy, by Olympus (1 mentions)
4 protocols using mito sox assay kit
1
NGR1 Neuroprotection and Oxidative Stress
2
Mitochondrial ROS Quantification by Flow Cytometry
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According to the manufacturer’s instructions, the mitochondrial ROS accumulation was performed with a MitoSOXassay kit (Invitrogen). In brief, isolated and cultured primary chondrocytes were incubated with 5 mM MitoSOX probe for 20 min at 37 °C in the dark. After a brief trypsinization with Trypsin-EDTA (0.05%) at 37 °C, cells were washed twice with FACS buffer (1× DPBS + 5% FBS), and flow cytometry was performed with a flow cytometer (Coulter-XL). EXPO32 ADC software was used for analysis.
3
Quantifying Intracellular and Mitochondrial ROS
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The intracellular ROS in C2C12 cells was measured by using a ROS Assay Kit (Beyotime, China). 2’, DCFH-DA, which is the ROS Assay Kit principal component, is oxidized easily to fluorescent dichlorofluorescein by cellular ROS. Therefore, intracellular ROS levels were detected. The mitochondrial ROS in C2C12 cells were detected by a Mito-Sox Assay Kit (Invitrogen, USA). According to the manufacturer’s protocol, C2C12 cells were washed with serum-free medium 3 times and treated with DCFH-DA and Mito-Sox for 20 mins respectively. After washing, C2C12 cells were stained with Hoechst (Beyotime, China) for 5 mins and then intracellular ROS was observed by fluorescence microscopy (Olympus, Japan) and mitochondrial ROS was observed by confocal laser scanning fluorescence microscopy (ZEISS, LSM800, Germany). The relative fluorescence intensive was measured by ImageJ software (NIH, USA).
4
Measuring Intracellular and Mitochondrial ROS
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The intracellular ROS in C2C12 cells was measured by using a ROS Assay Kit (Beyotime, China). 2', 7'-dichlorofluorescein-diacetate (DCFH-DA), which is the ROS Assay Kit principal component, is oxidized easily to fluorescent dichlorofluorescein by cellular ROS. Therefore, intracellular ROS levels were detected. The mitochondrial ROS in C2C12 cells were detected by a Mito-Sox Assay Kit (Invitrogen, USA). According to the manufacturer's protocol, C2C12 cells in different groups (Lv-vector, PA+Lv-vector, and PA+Lv-Gm20743) were washed with serum-free medium 3 times and treated with DCFH-DA and Mito-Sox for 20 mins respectively. After washing, C2C12 cells were stained with Hoechst (Beyotime, China) for 5 mins and then intracellular ROS was observed by fluorescence microscopy (Olympus, Japan) and mitochondrial ROS was observed by confocal laser scanning fluorescence microscopy (ZEISS, LSM800, Germany). The relative fluorescence intensive was measured by ImageJ software (NIH, USA).
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