MitoSOX Red
目录号 : GC68230MitoSOX Red是一种靶向线粒体的活细胞荧光探针，最大激发光/发射光为510/580 nm。
Cas No.:1003197-00-9
Sample solution is provided at 25 µL, 10mM.
Quality Control & SDS
- View current batch:
- Purity: >98.00%
- COA (Certificate Of Analysis)
- SDS (Safety Data Sheet)
- Datasheet
本方案仅提供一个指导，请根据您的具体需要进行修改。
1、制备MitoSOX Red染色液
（1）本产品以5mM的DMSO储存液形式提供，建议在首次使用前对产品进行离心，分装后在管内充氮气，于-20℃或-80℃避光保存。
（2）染料工作液：用合适的缓冲液(如:无血清培养基或PBS)稀释储存液，配制浓度为1-10μM的MitoSOX Red工作液。
注意: 请根据实际情况调整及优化工作液浓度，现用现配。
2、细胞悬浮染色(以6孔板为例)
（1）悬浮细胞经1000g离心3-5min。弃去上清液，使用PBS清洗两次，每次5分钟。
（2）贴壁细胞使用PBS清洗两次，加入胰酶消化细胞，消化完成后经1000g离心3-5min。
（3）加入1mL染料工作液重悬细胞，室温避光孵育5-30min分钟，不同细胞最佳培养时间不同。
（4）孵育结束后，经1000g离心5分钟，去除上清液，加入PBS清洗2-3次，每次5分钟。
（5）使用无血清细胞培养基或PBS重悬细胞，通过荧光显微镜或流式细胞技术进行观察。
3、细胞贴壁染色
（1）在无菌盖玻片上培养贴壁细胞。
（2）从培养基中移走盖玻片，吸出过量的培养基，将盖玻片放在潮湿的环境中。
（3）从盖玻片的一角加入100μL染料工作液，轻轻晃动使染料均匀覆盖所有细胞，室温避光孵育5-30min分钟。
（4）吸弃染料工作液，使用培养液清洗盖玻片2~3次，每次5分钟。
4、显微镜检测：MitoSOX Red的最大激发光/发射光为396/610nm。MitoSOX Red染料也可以在510nm处激发，如果MitoSOX Red染料在396nm而非510nm处激发，可能是该染料对线粒体超氧化物的标记更具选择性。
注意事项：
（1）荧光染料均存在淬灭问题，请尽量注意避光，以减缓荧光淬灭。
（2）为了您的安全和健康，请穿实验服并戴一次性手套操作。
MitoSOX Red is a live cell fluorescent probe targeting mitochondria with maximum excitation/emission light of 510/580 nm. MitoSOX Red has cell membrane permeability and can quickly and selectively target mitochondria after entering cells. After entering mitochondria, MitoSOX Red is easily oxidized by superoxide and produces strong red fluorescence after combining with nucleic acids in mitochondria. MitoSOX Red can be used as a fluorescent indicator to specifically detect superoxide[1-2].
Dihydroethidium, also called hydroethidine (HE), can be oxidized by reactive species and subsequently binds to DNA to produce fluorescence. MitoSOX Red is a dihydroethidium derivative with a cationic triphenylphosphonium moiety. This positively charged probe rapidly accumulates in mitochondria and can therefore be used to monitor superoxide/ROS production within mitochondria by fluorometry, microscopy or flow cytometry[3]. For example, in NK cells, MitoSOX red can observe the distribution of mitochondrial ROS[1]. Confocal microscopy imaging shows that in H9c2 cells treated with paraquat, the fluorescence intensity of MitoSOX Red-labeled mitochondria increases significantly[4].
References:
[1] Jin F, Wu Z, Hu X, Zhang J, Gao Z, Han X, Qin J, Li C, Wang Y. The PI3K/Akt/GSK-3β/ROS/eIF2B pathway promotes breast cancer growth and metastasis via suppression of NK cell cytotoxicity and tumor cell susceptibility. Cancer Biol Med. 2019 Feb;16(1):38-54.
[2] Milliken A S, Nadtochiy S M, Brookes P S. Inhibiting succinate release worsens cardiac reperfusion injury by enhancing mitochondrial reactive oxygen species generation[J]. Journal of the American Heart Association, 2022, 11(13): e026135.
[3] Kauffman ME, Kauffman MK, Traore K, Zhu H, Trush MA, Jia Z, Li YR. MitoSOX-Based Flow Cytometry for Detecting Mitochondrial ROS. React Oxyg Species (Apex). 2016;2(5):361-370.
[4] Mukhopadhyay P, Rajesh M, Yoshihiro K, et al. Simple quantitative detection of mitochondrial superoxide production in live cells[J]. Biochemical and biophysical research communications, 2007, 358(1): 203-208.
MitoSOX Red是一种靶向线粒体的活细胞荧光探针，最大激发光/发射光为510/580 nm。MitoSOX Red具有细胞膜渗透性，进入细胞后能够快速且选择性地靶向线粒体，进入线粒体后MitoSOX Red易被超氧化物氧化，与线粒体内核酸结合后，产生强烈的红色荧光。MitoSOX Red可以作为荧光指示剂，特异性检测超氧化物[1-2]。
二氢乙锭，也称为氢化乙锭 (HE)，可以被活性物质氧化，随后与DNA结合产生荧光。MitoSOX Red是带有阳离子三苯基鏻部分的二氢乙锭衍生物。这种带正电的探针在线粒体中快速积累，因此可用于通过荧光测定、显微镜或流式细胞术检测线粒体内超氧化物/ROS的产生[3]，例如，在NK细胞中，MitoSOX Red可以观察线粒体ROS的分布[1]；共聚焦显微成像显示，在用百草枯处理的 H9c2 细胞中，MitoSOX Red标记的线粒体荧光强度显著增加[4]。
| Cas No. | 1003197-00-9 | SDF | Download SDF |
| 分子式 | C43H43IN3P | 分子量 | 759.7 |
| 溶解度 | DMSO : 150 mg/mL (197.45 mM; Need ultrasonic) | 储存条件 | Store at -20°C,protect from light,stored under nitrogen |
| General tips | 请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。 储备液的保存方式和期限：-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。 为了提高溶解度，请将管子加热至37℃，然后在超声波浴中震荡一段时间。 |
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| Shipping Condition | 评估样品解决方案：配备蓝冰进行发货。所有其他可用尺寸：配备RT，或根据请求配备蓝冰。 | ||
| 制备储备液 | |||
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1 mg | 5 mg | 10 mg |
| 1 mM | 1.3163 mL | 6.5815 mL | 13.1631 mL |
| 5 mM | 0.2633 mL | 1.3163 mL | 2.6326 mL |
| 10 mM | 0.1316 mL | 0.6582 mL | 1.3163 mL |
| 第一步：请输入基本实验信息（考虑到实验过程中的损耗，建议多配一只动物的药量） | ||||||||||
| 给药剂量 | mg/kg |  |
动物平均体重 | g | |
每只动物给药体积 | ul | |
动物数量 | 只 |
| 第二步：请输入动物体内配方组成（配方适用于不溶于水的药物；不同批次药物配方比例不同，请联系GLPBIO为您提供正确的澄清溶液配方） | ||||||||||
| % DMSO % % Tween 80 % saline | ||||||||||
| 计算重置 | ||||||||||
计算结果:
工作液浓度： mg/ml；
DMSO母液配制方法： mg 药物溶于 μL DMSO溶液（母液浓度 mg/mL，
体内配方配制方法：取 μL DMSO母液，加入 μL PEG300，混匀澄清后加入μL Tween 80，混匀澄清后加入 μL saline，混匀澄清。
1. 首先保证母液是澄清的；
2.
一定要按照顺序依次将溶剂加入，进行下一步操作之前必须保证上一步操作得到的是澄清的溶液，可采用涡旋、超声或水浴加热等物理方法助溶。
3. 以上所有助溶剂都可在 GlpBio 网站选购。
Roles of TRPA1 and TRPV1 in cigarette smoke -induced airway epithelial cell injury model
Free Radic Biol Med 2019 Apr;134:229-238.PMID:30639616DOI:10.1016/j.freeradbiomed.2019.01.004.
Transient receptor potential protein (TRP) ion channels TRPA1 and TRPV1 may be important in mediating airway tissue injury and inflammation. This study was designed to clarify the role of TRPA1 and TRPV1 channels in cigarette smoke extract (CSE)-induced damage to bronchial and alveolar epithelial cells. Alveolar epithelial (A549) cells and bronchial epithelial (Beas-2B) cells were treated with CSE in the presence and absence of a TRPA1 inhibitor (100 μM, A967079), a TRPV1 inhibitor (100 μM, AMG9810) or both. DCFH-DA and MitoSOX Red probes were used to assay intracellular and mitochondrial oxidative stress, respectively. The mRNA levels of inflammatory mediators (IL-1β, IL-8, IL-18, IL-33) and antioxidants (HO-1, NQO1, MnSOD, catalase) and the protein expression levels of mitochondrial and inflammasome factors (MFN2, OPA1, DRP1, MFF, NLRP3,caspase-1) were respectively detected by RT-PCR and Western Blot. The results were validated in TRPA1 shRNA and TRPV1 shRNA cells. In both cell types, 10% CSE increased intracellular and mitochondrial oxidative stress, induced Ca2+ influx, increased inflammatory gene expression, reduced antioxidant gene expression and inhibited the activities of mitochondrial respiratory chain (MRC) complexes. 10% CSE increased the expression of mitochondrial fission proteins (MFF and DRP1), Caspase-1 and NLRP3 protein expression and decreased that of mitochondrial fusion proteins (MFN2 and OPA1). Both inhibitors and gene-knockout of TRPA1 and TRPV1 reduced oxidative stress, blocked Ca2+ influx, and inhibited inflammatory and increased antioxidant gene expression. They also prevented the changes in mitochondrial fission and fusion proteins and in MRC complexes activities induced by CSE. Both TRPA1 and TRPV1 mediate CSE-induced damage of bronchial and alveolar epithelial cells via modulation of oxidative stress, inflammation and mitochondrial damage and their inhibition should be considered as potential therapy for COPD.
Mitochondrial DNA leakage induces odontoblast inflammation via the cGAS-STING pathway
Cell Commun Signal 2021 May 20;19(1):58.PMID:34016129DOI:10.1186/s12964-021-00738-7.
Background: Mitochondrial DNA (mtDNA) is a vital driver of inflammation when it leaks from damaged mitochondria into the cytosol. mtDNA stress may contribute to cyclic GMP-AMP synthase (cGAS) stimulator of interferon genes (STING) pathway activation in infectious diseases. Odontoblasts are the first cells challenged by cariogenic bacteria and involved in maintenance of the pulp immune and inflammatory responses to dentine-invading pathogens. In this study, we investigated that mtDNA as an important inflammatory driver participated in defending against bacterial invasion via cGAS-STING pathway in odontoblasts. Methods: The normal tissues, caries tissues and pulpitis tissues were measured by western blotting and immunohistochemical staining. Pulpitis model was built in vitro to evaluated the effect of the cGAS-STING pathway in odontoblast-like cell line (mDPC6T) under inflammation. Western blot and real-time PCR were performed to detect the expression of cGAS-STING pathway and pro-inflammatory cytokines. The mitochondrial function was evaluated reactive oxygen species (ROS) generated by mitochondria using MitoSOX Red dye staining. Cytosolic DNA was assessed by immunofluorescent staining and real-time PCR in mDPC6T cells after LPS stimulation. Furthermore, mDPC6T cells were treated with ethidium bromide (EtBr) to deplete mtDNA or transfected with isolated mtDNA. The expression of cGAS-STING pathway and pro-inflammatory cytokines were measured. Results: The high expression of cGAS and STING in caries and pulpitis tissues in patients, which was associated with inflammatory progression. The cGAS-STING pathway was activated in inflamed mDPC6T. STING knockdown inhibited the nuclear import of p65 and IRF3 and restricted the secretion of the inflammatory cytokines CXCL10 and IL-6 induced by LPS. LPS caused mitochondrial damage in mDPC6T, which promoted mtDNA leakage into the cytosol. Depletion of mtDNA inhibited the cGAS-STING pathway and nuclear translocation of p65 and IRF3. Moreover, repletion of mtDNA rescued the inflammatory response, which was inhibited by STING knockdown. Conclusion: Our study systematically identified a novel mechanism of LPS-induced odontoblast inflammation, which involved mtDNA leakage from damaged mitochondria into the cytosol stimulating the cGAS-STING pathway and the inflammatory cytokines IL-6 and CXCL10 secretion. The mtDNA-cGAS-STING axis could be a potent therapeutic target to prevent severe bacterial inflammation in pulpitis. Video Abstract.
Hydroethidine- and MitoSOX-derived red fluorescence is not a reliable indicator of intracellular superoxide formation: another inconvenient truth
Free Radic Biol Med 2010 Apr 15;48(8):983-1001.PMID:20116425DOI:10.1016/j.freeradbiomed.2010.01.028.
Hydroethidine (HE; or dihydroethidium) is the most popular fluorogenic probe used for detecting intracellular superoxide radical anion. The reaction between superoxide and HE generates a highly specific red fluorescent product, 2-hydroxyethidium (2-OH-E(+)). In biological systems, another red fluorescent product, ethidium, is also formed, usually at a much higher concentration than 2-OH-E(+). In this article, we review the methods to selectively detect the superoxide-specific product (2-OH-E(+)) and the factors affecting its levels in cellular and biological systems. The most important conclusion of this review is that it is nearly impossible to assess the intracellular levels of the superoxide-specific product, 2-OH-E(+), using confocal microscopy or other fluorescence-based microscopic assays and that it is essential to measure by HPLC the intracellular HE and other oxidation products of HE, in addition to 2-OH-E(+), to fully understand the origin of red fluorescence. The chemical reactivity of mitochondria-targeted hydroethidine (Mito-HE, MitoSOX Red) with superoxide is similar to the reactivity of HE with superoxide, and therefore, all of the limitations attributed to the HE assay are applicable to Mito-HE (or MitoSOX) as well.
Detection of Mitochondrial Mass, Damage, and Reactive Oxygen Species by Flow Cytometry
Cold Spring Harb Protoc 2015 Sep 1;2015(9):pdb.prot086298.PMID:26330624DOI:10.1101/pdb.prot086298.
The reagents and procedures highlighted here will give the investigators an indication of the health status and volume of mitochondria in primary cells and cell lines through the use of a number of cell-permeable dyes. Mitochondrial volume can be monitored by using the probe MitoTracker Green FM. This reagent labels mitochondria in a manner that is independent of the membrane potential, therefore providing a readout relating purely to the mitochondrial mass of the cell. In contrast, MitoTracker Red CMXRos, tetramethylrhodamine methyl ester, and 10-N-nonyl acridine orange label mitochondria in a manner dependent on the membrane potential, thus giving an indication of mitochondrial stress. Using MitoSOX Red, it is also possible to analyze the production of the mitochondrial superoxide anion. Like the MitoTracker probes, MitoSOX Red is taken up passively by cells. In the mitochondria, the probe is oxidized by superoxide, resulting in the emission of red fluorescence.
Curcumin attenuates MSU crystal-induced inflammation by inhibiting the degradation of IκBα and blocking mitochondrial damage
Arthritis Res Ther 2019 Aug 27;21(1):193.PMID:31455356DOI:10.1186/s13075-019-1974-z.
Background: Gouty arthritis is characterized by the deposition of monosodium urate (MSU) within synovial joints and tissues due to increased urate concentrations. In this study, we explored the effect of the natural compound curcumin on the MSU crystal-stimulated inflammatory response. Methods: THP-1-derived macrophages and murine RAW264.7 macrophages were pretreated with curcumin for 1 h and then stimulated with MSU suspensions for 24 h. The protein level of TLR4, MyD88, and IκBα, the activation of the NF-κB signaling pathway, the expression of the NF-κB downstream inflammatory cytokines, and the activity of NLRP3 inflammasome were measured by western blotting and ELISA. THP-1 and RAW264.7 cells were loaded with MitoTracker Green to measure mitochondrial content, and MitoTracker Red to detect mitochondrial membrane potential. To measure mitochondrial reactive oxygen species (ROS) levels, cells were loaded with MitoSOX Red, which is a mitochondrial superoxide indicator. The effects of curcumin on mouse models of acute gout induced by the injection of MSU crystals into the footpad and synovial space of the ankle, paw and ankle joint swelling, lymphocyte infiltration, and MPO activity were evaluated. Results: Curcumin treatment markedly inhibited the degradation of IκBα, the activation of NF-κB signaling pathway, and the expression levels of the NF-κB downstream inflammatory genes such as IL-1β, IL-6, TNF-α, COX-2, and PGE2 in the MSU-stimulated THP-1-derived macrophages. Curcumin administration protected THP-1 and RAW264.7 cells from MSU induced mitochondrial damage through preventing mitochondrial membrane potential reduction, decreasing mitochondria ROS, and then inhibited the activity of NLRP3 inflammasome. Intraperitoneal administration of curcumin alleviated MSU crystal-induced paw and ankle joint swelling, inflammatory cell infiltration, and MPO activity in mouse models of acute gout. These results correlated with the inhibition of the degradation of IκBα, the phosphorylation levels of NF-κB subunits (p65 and p50), and the activity of NLRP3 inflammasome. Conclusion: Curcumin administration effectively alleviated MSU-induced inflammation by suppressing the degradation of IκBα, the activation NF-κB signaling pathway, the damage of mitochondria, and the activity of NLRP3 inflammasome. Our results provide a new strategy in which curcumin therapy may be helpful in the prevention of acute episodes of gout.