NucPE1 (Nuclear Peroxy Emerald 1)
(Synonyms: Nuclear Peroxy Emerald 1) 目录号 : GC30066
NucPE1 (Nuclear Peroxy Emerald 1) (Nuclear Peroxy Emerald 1) 是一种核定位荧光过氧化氢，可特异性定位于细胞核，而无需附加靶向部分。
Cas No.:1404091-23-1
Sample solution is provided at 25 µL, 10mM.
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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NucPE1 (Nuclear Peroxy Emerald 1)染色液
(1) 制备染料储存液: 使用DMSO将NucPE1溶解成5-10mM的储存液。
注意:未使用的储存液建议分装后于-4℃或-20℃避光保存，避免反复冻融。
(2) 制备染料工作液:用合适的缓冲液(如:无血清培养基，HBSS或PBS)稀释储存液，配制浓度为1-10μM的NucPE1工作液。
注意: 请根据实际情况调整染料工作液浓度，现用现配。
2、细胞贴壁染色
(1)使用荧光染料的前一天进行细胞铺板。
(2)实验药物/处理处理细胞后，使用PBS漂洗2-3次。
(3)加入预热的NucPE1染料工作液，37°C避光孵育15-30分钟。不同细胞最佳孵育时间不同，请根据具体实验需求自行摸索。
(4) 孵育结束后，使用PBS洗涤2-3次，每次5分钟。
(5) 使用预温的无血清细胞培养基或PBS覆盖细胞（保持细胞湿润）。
3、细胞悬浮染色
(1)悬浮细胞：悬浮细胞经4°C、1000g离心3-5分钟，弃去上清液，用PBS清洗两次，每次5分钟。
(2)贴壁细胞：使用PBS冲洗2-3次后，使用胰蛋白酶消化细胞，4°C、1000g离心3-5分钟，弃去上清液。
(3) 使用0.5-1mL工作液重悬约1 x 106个细胞，37°C避光孵育15-30分钟。不同细胞最佳孵育时间不同，请根据具体实验需求自行摸索。
4、荧光检测：使用共聚焦显微镜或流式细胞技术进行分析[1]。NucPE1探针的实时成像使用氩激光在488nm处激发进行，并使用约520nm的META检测器收集发射；当使用NucPE1和PO1进行多次染色时，应用多跟踪扫描模式采集图像[2]。
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。
References:
[1]. Stanicka J, et al. NADPH oxidase-generated hydrogen peroxide induces DNA damage in mutant FLT3-expressing leukemia cells. J Biol Chem. 2015 Apr 10;290(15):9348-61.
[2]. Dickinson BC, et al. A nuclear-localized fluorescent hydrogen peroxide probe for monitoring sirtuin-mediated oxidative stress responses in vivo. Chem Biol. 2011 Aug 26;18(8):943-8.
NucPE1 (Nuclear Peroxy Emerald 1) is a nuclear-localized fluorescent hydrogen peroxide that is specifically localized to cellular nuclei without appended targeting moieties.
NucPE1 features two major visible region absorptions (λabs=468 nm, ε=27,300 M-1cm-1; λabs=490 nm, ε=26,000 M-1cm-1) and a weak emission (λem=530 nm, Φ=0.117). Reaction of NucPE1 with H2O2 triggers a fluorescence increase upon its conversion to fluorophore NucPE1, which possesses one major absorption band at 505 nm (ε=19,100 M-1cm-1) with enhanced emission (λem=530 nm, Φ=0.626). NucPE1 selectively accumulates in the nuclei of a variety of mammalian cell lines as well as in whole model organisms like C. clegans, where it can respond to subcellular changes in H2O2 fluxes[1].
NucPE1 maintains the ability to selectively target nuclei in vivo. NucPE1 imaging reveals a reduction in nuclear H2O2 levels in worms overexpressing sir-2.1 compared to wildtype congeners, supporting a link between this longevity-promoting sirtuin protein and enhanced regulation of nuclear ROS pools[1].
[1]. Stanicka J, et al. NADPH oxidase-generated hydrogen peroxide induces DNA damage in mutant FLT3-expressing leukemia cells. J Biol Chem. 2015 Apr 10;290(15):9348-61. [2]. Dickinson BC, et al. A nuclear-localized fluorescent hydrogen peroxide probe for monitoring sirtuin-mediated oxidative stress responses in vivo. Chem Biol. 2011 Aug 26;18(8):943-8.
| Cas No. | 1404091-23-1 | SDF | |
| 别名 | Nuclear Peroxy Emerald 1 | ||
| Canonical SMILES | O=C1OC2(C3=C(OC4=C2C=CC(B5OC(C)(C)C(C)(C)O5)=C4)C=C(NCC)C(C)=C3)C6=C1C=CC=C6 | ||
| 分子式 | C29H30BNO5 | 分子量 | 483.36 |
| 溶解度 | DMSO : 25 mg/mL (51.72 mM) | 储存条件 | Store at -20°C |
| 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 | 2.0689 mL | 10.3443 mL | 20.6885 mL |
| 5 mM | 0.4138 mL | 2.0689 mL | 4.1377 mL |
| 10 mM | 0.2069 mL | 1.0344 mL | 2.0689 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。
A nuclear-localized fluorescent hydrogen peroxide probe for monitoring sirtuin-mediated oxidative stress responses in vivo
Hydrogen peroxide (H(2)O(2)) can serve as a beneficial signaling agent or toxin depending on its concentration and location within a cell or organism. Methods to measure the localized accumulation of H(2)O(2) in living specimens remain limited. Motivated to meet this need, we have developed a nuclear-localized fluorescent probe for H(2)O(2), Nuclear Peroxy Emerald 1 (NucPE1), to selectively interrogate ROS fluxes within this sensitive organelle. NucPE1 selectively accumulates in the nuclei of a variety of mammalian cell lines as well as in whole model organisms like Caenorhabditis elegans, where it can respond to subcellular changes in H(2)O(2) fluxes. Moreover, in vivo NucPE1 imaging reveals a reduction in nuclear H(2)O(2) levels in worms overexpressing sir-2.1 compared with wild-type congeners, supporting a link between this longevity-promoting sirtuin protein and enhanced regulation of nuclear ROS pools.
NADPH oxidase-generated hydrogen peroxide induces DNA damage in mutant FLT3-expressing leukemia cells
Internal tandem duplication of the FMS-like tyrosine kinase (FLT3-ITD) receptor is present in 20% of acute myeloid leukemia (AML) patients and it has been associated with an aggressive AML phenotype. FLT3-ITD expressing cell lines have been shown to generate increased levels of reactive oxygen species (ROS) and DNA double strand breaks (DSBs). However, the molecular basis of how FLT3-ITD-driven ROS leads to the aggressive form of AML is not clearly understood. Our group has previously reported that inhibition of FLT3-ITD signaling results in post-translational down-regulation of p22(phox), a small membrane-bound subunit of the NADPH oxidase (NOX) complex. Here we demonstrated that 32D cells, a myeloblast-like cell line transfected with FLT3-ITD, have a higher protein level of p22(phox) and p22(phox)-interacting NOX isoforms than 32D cells transfected with the wild type FLT3 receptor (FLT3-WT). The inhibition of NOX proteins, p22(phox), and NOX protein knockdowns caused a reduction in ROS, as measured with a hydrogen peroxide (H2O2)-specific dye, peroxy orange 1 (PO1), and nuclear H2O2, as measured with nuclear peroxy emerald 1 (NucPE1). These reductions in the level of H2O2 following the NOX knockdowns were accompanied by a decrease in the number of DNA DSBs. We showed that 32D cells that express FLT3-ITD have a higher level of both oxidized DNA and DNA DSBs than their wild type counterparts. We also observed that NOX4 and p22(phox) localize to the nuclear membrane in MV4-11 cells expressing FLT3-ITD. Taken together these data indicate that NOX and p22(phox) mediate the ROS production from FLT3-ITD that signal to the nucleus causing genomic instability.