M36008

Manufactured by Thermo Fisher Scientific

Sourced in United States

The M36008 is a laboratory instrument designed for sample preparation and analysis. Its core function is to facilitate the extraction, separation, and detection of analytes from various sample types. The product specifications and capabilities are available upon request.

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MitoSOX Red (M36008), (11 citations) MitoSOX Red mitochondrial superoxide indicator (M36008) (11 citations) MitoSox M36008 (2 citations)

43 protocols using m36008

Quantifying Oxidative Stress in Cardiomyocytes

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The total cellular and mitochondrial ROS contents in frozen heart sections were evaluated by DHE staining (10 μM, S0063, Beyotime, China) and staining with the fluorescent probe MitoSOX (100 mM, M36008, Thermo Fisher Scientific, USA), respectively, as previously described [21 (link)]. Images were captured with a confocal laser scanning microscope (Nikon, Japan) and analysed with ImageJ software (NIH, USA).
Total cellular ROS in NCMs were detected by a ROS/Superoxide Detection Assay Kit (Ab139476, Abcam, USA) as previously described via a microplate reader [22 (link)]. The mitochondrial ROS in NCMs were detected by staining with the fluorescent probe MitoSOX (100 mM, M36008, Thermo Fisher Scientific, USA) following the manufacturers’ protocols via flow cytometry analysis with a BD FACS Aria II flow cytometer.
The Total Glutathione Peroxidase (GPx) Assay Kit with NADPH (S0058, Beyotime, China) and Lipid Peroxidation Malondialdehyde (MDA) Assay Kit (Ab118970, Abcam, USA) were used according to the manufacturer’s instructions to detect cardiomyocyte GPx activity and MDA levels and further assess oxidative stress levels.

Yang R., Zhang X., Zhang Y., Wang Y., Li M., Meng Y., Wang J., Wen X., Yu J, & Chang P. (2023). Grpel2 maintains cardiomyocyte survival in diabetic cardiomyopathy through DLST-mediated mitochondrial dysfunction: a proof-of-concept study. Journal of Translational Medicine, 21, 200.

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Oxidative Stress Measurement in Rat Hippocampus

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Rats were anesthetized and perfused with precooled phosphate buffered saline (PBS) solution. The hippocampus was then separated and prepared into single cell suspensions using mechanical digestion and the trypsin-EDTA solution (T1320, Solarbio, China). The cells were quantified and incubated in H2DCFDA (15 μM, HY-D0940, MCE, USA) or MitoSOX (1 μM, M36008, Thermo, USA) at 37°C for 30 minutes. Finally, the cells were washed with precooled PBS solution and transferred into a fluorescence-activated cell sorting (FACS) tube. The ROS production was measured in a flow cytometer (BD, USA) and analyzed by the FlowJo software (TreeStar Inc.).

Hu X., Wu L., Wen Y., Liu J., Li H., Zhang Y., Wang Z., Ding J., Zeng Z, & Xia H. (2022). Hippocampal Mitochondrial Abnormalities Induced the Dendritic Complexity Reduction and Cognitive Decline in a Rat Model of Spinal Cord Injury. Oxidative Medicine and Cellular Longevity, 2022, 9253916.

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Mitochondrial ROS Detection via MitoSOX

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MitoSOX Red mitochondrial superoxide indicator was utilized for the detection of mitochondrial ROS (M36008, Thermo Fisher Scientific, Waltham, MA, USA). The dye is selectively oxidized by respiratory chain‐derived superoxides, emitting red fluorescence (~ 580 nm). Cells were stained with 0.2 µM MitoSOX in pre‐warmed hanks balanced salt solution (HBSS), incubated in the dark at 37°C and immediately analyzed using a FACS Canto II flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).

Hirschberger S., Strauß G., Effinger D., Marstaller X., Ferstl A., Müller M.B., Wu T., Hübner M., Rahmel T., Mascolo H., Exner N., Heß J., Kreth F.W., Unger K, & Kreth S. (2021). Very‐low‐carbohydrate diet enhances human T‐cell immunity through immunometabolic reprogramming. EMBO Molecular Medicine, 13(8), e14323.

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Mitochondrial Function and Autophagy Assays

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Mitochondrial membrane potential was measured in PK1 cells by tetramethylrhodamine ethyl ester (TMRE) staining (50 nM for 20 min at 37 °C, Thermo-Fisher T669, Waltham, MA, USA) [54 (link)], and mitochondrial ROS production was assessed by MitoSOX (2 μM for 30 min at 37 °C, Thermo-Fisher M36008) [25 (link)].
Protein concentration was measured with Bradford Protein Assay. Specific antibodies against mTOR, ULK-1, ATG-5-12, LC3 I/II, p-AMPK, AMPK, p-AKT, AKT, p-S6, S6 and GADPH were used as mentioned above. The density of each band was analyzed byAlphaView SA software (Cell Biosciences, Santa Clara, CA, USA).

Hong S., Ghandriz R., Siddiqi S., Zhu X.Y., Saadiq I.M., Jordan K.L., Tang H., Ali K.A., Lerman A., Eirin A, & Lerman L.O. (2022). Effects of Elamipretide on Autophagy in Renal Cells of Pigs with Metabolic Syndrome. Cells, 11(18), 2891.

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Quantifying Intracellular ROS in ASCs

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Total intracellular or mitochondrial reactive oxygen species (ROS) were examined in ASCs using DCFH-DA (D6883, Sigma-Aldrich) or MitoSOX (M36008, Thermo Fisher, Waltham, MA), respectively. Briefly, ASCs were cultured for 9 h of attachment, followed by incubation with 10 μM DCFH-DA for 30 min or 5 μM MitoSOX for 10 min at 37 °C. Fluorescent signals were detected by flow cytometry (cytoFLEX, Beckman-Couler, Bria, CA). In some experiments, ASCs were cultured in 96-well plate (1 × 10⁴/well) for ROS fluorescence imaging using Opera Phenix Plus High-Content Screening System (PerkinElmer). For ROS elimination, ASCs were treated with 5 mM N-acetylcysteine (NAC,A7250, Sigma-Aldrich) or 5 μM Mito-TEMPO (SML0737, Sigma-Aldrich) for 12 h before related assays.

Zhou Z., Zhang H., Tao Y., Zang J., Zhao J., Li H., Wang Y., Wang T., Zhao H., Wang F., Guo C., Zhu F., Mao H., Liu F., Zhang L, & Wang Q. (2023). FGF21 alleviates adipose stem cell senescence via CD90 glycosylation-dependent glucose influx in remodeling healthy white adipose tissue. Redox Biology, 67, 102877.

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Evaluation of Oxidative Stress-Induced Damage

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Immunohistochemistry staining were used to evaluate the oxidative stress-induced DNA damage (8-OHdG) and mitochondrial superoxide (Mitosox) in the brain. Freshly prepared frozen 10μm brain slices were placed on the microscope slides (Genesee Scientific, CA, USA). The slides were first immersed in the pH 6.0 antigen retrieval solution and heated in microwave oven for 15 min to expose the antigen. Secondly, the sections were blocked with endogenous peroxidase for 10 min using 3% H2O2 followed by the incubation of 8-OHdG antibody (1:200, ab62623, Abcam, MA, USA) or Mitosox antibody (1:1000, M36008, Thermo Fisher, CA, USA) at room temperature. The slides were observed under the microscope under magnification of 200x for Mitosox staining or 400x for 8-OHdG. The fluorescence intensity was averaged from four randomly selected brain regions within ipsilateral entorhinal cortex using ImageJ 1.52p (National Institutes of Health, MD, USA).

Huang Y., Guo Y., Huang L., Fang Y., Li D., Liu R., Lu Q., Ren R., Tang L., Lian L., Hu Y., Tang J., Chen G, & Zhang J.H. (2021). Kisspeptin-54 attenuates oxidative stress and neuronal apoptosis in early brain injury after subarachnoid hemorrhage in rats via GPR54/ARRB2/AKT/GSK3β signaling pathway. Free radical biology & medicine, 171, 99-111.

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Quantifying Mitochondrial ROS in PASMCs

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mROS were measured using a highly selective molecular probe, MitoSOX^TM Red mitochondrial superoxide indicator, for live-cell imaging (M36008, ThermoFisher Scientific). Briefly, PASMCs (1.0 × 10⁵) were seeded on chamber slides. When PASMCs grew to 50% confluence, all chambers were exposed to normoxia (21% O₂) and hypoxia (5% O₂) with or without MitoQ (0.5 μM) incubation. After 12 h, they were stained with MitoSOX^TM working solution (5 μM) within a hypoxic glove chamber for 10 min. Following washing, all chambers were observed immediately under confocal laser scanning microscope (Olympus FLUOVIEW FV 1000). The density of fluorescent signal for mROS was quantified using Image-Pro Plus.

Chen J., Zhang M., Liu Y., Zhao S., Wang Y., Wang M., Niu W., Jin F, & Li Z. (2022). Histone lactylation driven by mROS-mediated glycolytic shift promotes hypoxic pulmonary hypertension. Journal of Molecular Cell Biology, 14(12), mjac073.

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Quantifying Oxidative Stress in Brain Sections

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The 8-hydroxy-2′-deoxyguanosine (8OHdG) and Mitosox staining were used to assess to the oxidative-stress-induced DNA damage and mitochondrial superoxide level [25 (link)]. Briefly, the freshly prepared frozen 15 μm coronal brain sections were immersed in antigen retrieval solution (PH 6.0) and heated in a microwave oven for 15 min. Sections were mounted with 3% H₂O₂ for 10 min to block endogenous peroxidase. The 8OHdG antibody (ab62623, Abcam, Waltham, MA, USA) and Mitosox antibody (M36008, Thermo Fisher, Waltham, MA, USA) were incubated at room temperature. The cell nuclei were stained by 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich, St. Louis, MO, USA). Finally, the brain slices were observed using a fluorescence microscope (DMi8, Leica Microsystems, Wetzlar, Germany) under 400× or 200× magnification and averaged from four randomly selected regions within the ipsilateral entorhinal cortex.

Huang Y., Wu H., Hu Y., Zhou C., Wu J., Wu Y., Wang H., Lenahan C., Huang L., Nie S., Gao X, & Sun J. (2022). Puerarin Attenuates Oxidative Stress and Ferroptosis via AMPK/PGC1α/Nrf2 Pathway after Subarachnoid Hemorrhage in Rats. Antioxidants, 11(7), 1259.

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Mitochondrial Superoxide Measurement

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BUMPT cells were treated with cisplatin or/with sTim-3 protein (80 μg/mL) for 18 h. Then, BUMPT cells were stained with MitoSOX Red dye (an indicator of mitochondrial superoxide, 5 μM, M36008, Thermo Fisher Scientific, USA) for 10 min at 37 °C, and then were observed using a confocal microscope (NIKON A1R Storm).

Li P., Li X., Wu W., Hou M., Yin G., Wang Z., Du Z., Ma Y., Lou Q, & Wei Y. (2023). Tim-3 protects against cisplatin nephrotoxicity by inhibiting NF-κB-mediated inflammation. Cell Death Discovery, 9, 218.

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Mitochondrial Superoxide Evaluation in Osteoblast Differentiation

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Mitochondrial superoxide production was evaluated by the mitosox red compound (M36008, Thermo Fisher Scientific, USA). Oxidation of mitosox red by superoxide compounds present in mitochondria produces red fluorescence. In brief, we prepared a 5 mM stock solution of mitosox in DMSO. DPSCs (2 × 10⁴) were seeded in a 10 cm dish overnight. After 16 h, cells were treated with BGP + LAA for 14 days for osteoblast differentiation. Control cells were cultured for 14 days with a culture medium (α-MEM). After differentiation of cells or undifferentiated cells were washed with ice-cold 1 x PBS and incubated with 2 μM (working solution) of mitosox red for 30 min at 37 °C. Then, cells were harvested through scrapping and fluorescence intensity was measured using a plate reader (Synergy 2, BioTek Instruments, Inc, Winooski, USA) at a setting excitation of 510 nm and emission at 580 nm using 200 μl/well of lysate. Each experiment was performed at least three times and used triplicate for each assay.

Maity J., Deb M., Greene C, & Das H. (2020). KLF2 regulates dental pulp-derived stem cell differentiation through the induction of mitophagy and altering mitochondrial metabolism. Redox Biology, 36, 101622.

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