DAF-2
(Synonyms: 4,5-Diaminofluorescein) 目录号 : GC43372A fluorescent probe used for the detection of NO
Cas No.:205391-01-1
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．本产品以DMSO储存液形式提供，浓度为1 mM，不建议更换溶剂；
2．未使用的储存液建议分装后在-20℃或-80℃避光保存，避免反复冻融；
3．染料使用前请先短暂离心，以保证产品集中在管底；
4．BSA和酚红(phenol red)对DAF-2的检测有干扰，建议使用合适的缓冲液(如:无血清和酚红的培养基或PBS)稀释储存液，配制浓度为1-10μM的工作液；
5．稀释后的水溶性溶液存放不要超过一天，请根据实际情况调整工作液浓度，现用现配；
6．显微镜检测：DAF-2的最大激发光/发射光波长为485/538nm。
7．DAF-2不具细胞膜渗透性，可用于胞外或溶液体系内一氧化氮的直接检测；若需要做胞内一氧化氮的检测，可选择具细胞膜渗透性的DAF-2衍生物DAF-2 DA(GC30702)；
8．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。
为了您的安全和健康，请穿实验服并戴一次性手套操作。
DAF-2 is a sensitive fluorescent indicator commonly used for the detection of nitric oxide (NO).[1],[2] It reacts with NO in the presence of oxygen to yield the highly fluorescent triazolofluorescein (DAF-2T). Fluorescence is monitored using excitation and emission wavelengths of 485 and 538 nM, respectively.2 At neutral pH the detection limit for NO is 2-5 nM.
Reference:
[1]. Kojima, H., Sakurai, K., Kikuchi, K., et al. Development of a fluorescent indicator for nitric oxide based on the fluorescein chromophore. Chemical & Pharmaceutical Bulletin 46, 373-375 (1998).
[2]. Nakatsubo, N., Kojima, H., Kikuchi, K., et al. Direct evidence of nitric oxide production from bovine aortic endothelial cells using new fluorescence indicators: Diaminofluoresceins. FEBS Letters 427, 263-266 (1998).
| Cas No. | 205391-01-1 | SDF | |
| 别名 | 4,5-Diaminofluorescein | ||
| 化学名 | 2-(3,6-dihydroxy-4,5-diamino-9H-xanthen-9-yl)-benzoic acid | ||
| Canonical SMILES | OC1=CC2=C(C=C1)C3(C(C=C(N)C(N)=C4)=C4C(O3)=O)C5=CC=C(O)C=C5O2 | ||
| 分子式 | C20H14N2O5 | 分子量 | 362.3 |
| 溶解度 | A solution in DMSO | 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 | 2.7601 mL | 13.8007 mL | 27.6014 mL |
| 5 mM | 0.552 mL | 2.7601 mL | 5.5203 mL |
| 10 mM | 0.276 mL | 1.3801 mL | 2.7601 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。
DAF-16/FoxO in Caenorhabditis elegans and Its Role in Metabolic Remodeling
Cells 2020 Jan 2;9(1):109.PMID:31906434DOI:10.3390/cells9010109.
DAF-16, the only forkhead box transcription factors class O (FoxO) homolog in Caenorhabditis elegans, integrates signals from upstream pathways to elicit transcriptional changes in many genes involved in aging, development, stress, metabolism, and immunity. The major regulator of DAF-16 activity is the insulin/insulin-like growth factor 1 (IGF-1) signaling (IIS) pathway, reduction of which leads to lifespan extension in worms, flies, mice, and humans. In C. elegans DAF-2 mutants, reduced IIS leads to a heterochronic activation of a dauer survival program during adulthood. This program includes elevated antioxidant defense and a metabolic shift toward accumulation of carbohydrates (i.e., trehalose and glycogen) and triglycerides, and activation of the glyoxylate shunt, which could allow fat-to-carbohydrate conversion. The longevity of DAF-2 mutants seems to be partially supported by endogenous trehalose, a nonreducing disaccharide that mammals cannot synthesize, which points toward considerable differences in downstream mechanisms by which IIS regulates aging in distinct groups.
DAF-2, an insulin receptor-like gene that regulates longevity and diapause in Caenorhabditis elegans
Science 1997 Aug 15;277(5328):942-6.PMID:9252323DOI:10.1126/science.277.5328.942.
A C. elegans neurosecretory signaling system regulates whether animals enter the reproductive life cycle or arrest development at the long-lived dauer diapause stage. DAF-2, a key gene in the genetic pathway that mediates this endocrine signaling, encodes an insulin receptor family member. Decreases in DAF-2 signaling induce metabolic and developmental changes, as in mammalian metabolic control by the insulin receptor. Decreased DAF-2 signaling also causes an increase in life-span. Life-span regulation by insulin-like metabolic control is analogous to mammalian longevity enhancement induced by caloric restriction, suggesting a general link between metabolism, diapause, and longevity.
Sulforaphane promotes C. elegans longevity and healthspan via DAF-16/DAF-2 insulin/IGF-1 signaling
Aging (Albany NY) 2021 Jan 20;13(2):1649-1670.PMID:33471780DOI:10.18632/aging.202512.
The broccoli-derived isothiocyanate sulforaphane inhibits inflammation, oxidative stress and cancer, but its effect on healthspan and longevity are unclear. We used the C. elegans nematode model and fed the wildtype and 9 mutant strains ±sulforaphane. The lifespan, phenotype, pharyngeal pumping, mobility, lipofuscin accumulation, and RNA and protein expression of the nematodes were assessed by using Kaplan-Meier survival analysis, in vivo live imaging, fluorescence microscopy, and qRT-PCR. Sulforaphane increased the lifespan and promoted a health-related phenotype by increasing mobility, appetite and food intake and reducing lipofuscin accumulation. Mechanistically, sulforaphane inhibited DAF-2-mediated insulin/insulin-like growth factor signaling and its downstream targets AGE-1, AKT-1/AKT-2. This was associated with increased nuclear translocation of the FOXO transcription factor homolog DAF-16. In turn, the target genes sod-3, mtl-1 and gst-4, known to enhance stress resistance and lifespan, were upregulated. These results indicate that sulforaphane prolongs the lifespan and healthspan of C. elegans through insulin/IGF-1 signaling. Our results provide the basis for a nutritional sulforaphane-enriched strategy for the promotion of healthy aging and disease prevention.
DAF-2/insulin IGF-1 receptor regulates motility during aging by integrating opposite signaling from muscle and neuronal tissues
Aging Cell 2022 Aug;21(8):e13660.PMID:35808897DOI:10.1111/acel.13660.
During aging, preservation of locomotion is generally considered an indicator of sustained good health, in elderlies and in animal models. In Caenorhabditis elegans, mutants of the insulin-IGF-1 receptor DAF2/IIRc represent a paradigm of healthy aging, as their increased lifespan is accompanied by a delay in age-related loss of motility. Here, we investigated the DAF-2/IIRc-dependent relationship between longevity and motility using an auxin-inducible degron to trigger tissue-specific degradation of endogenous DAF-2/IIRc. As previously reported, inactivation of DAF-2/IIRc in neurons or intestine was sufficient to extend the lifespan of worms, whereas depletion in epidermis, germline, or muscle was not. However, neither intestinal nor neuronal depletion of DAF-2/IIRc prevented the age-related loss of motility. In 1-day-old adults, DAF-2/IIRc depletion in neurons reduced motility in a DAF-16/FOXO dependent manner, while muscle depletion had no effect. By contrast, DAF-2 depletion in the muscle of middle-age animals improved their motility independently of DAF-16/FOXO but required UNC-120/SRF. Yet, neuronal or muscle DAF-2/IIRc depletion both preserved the mitochondria network in aging muscle. Overall, these results show that the motility pattern of DAF-2 mutants is determined by the sequential and opposing impact of neurons and muscle tissues and can be dissociated from the regulation of the lifespan. This work also provides the characterization of a versatile tool to analyze the tissue-specific contribution of insulin-like signaling in integrated phenotypes at the whole organism level.
H3K9me1/2 methylation limits the lifespan of DAF-2 mutants in C. elegans
Elife 2022 Sep 20;11:e74812.PMID:36125117DOI:10.7554/eLife.74812.
Histone methylation plays crucial roles in the development, gene regulation, and maintenance of stem cell pluripotency in mammals. Recent work shows that histone methylation is associated with aging, yet the underlying mechanism remains unclear. In this work, we identified a class of putative histone 3 lysine 9 mono/dimethyltransferase genes (met-2, set-6, set-19, set-20, set-21, set-32, and set-33), mutations in which induce synergistic lifespan extension in the long-lived DAF-2 (insulin growth factor 1 [IGF-1] receptor) mutant in Caenorhabditis elegans. These putative histone methyltransferase plus DAF-2 double mutants not only exhibited an average lifespan nearly three times that of wild-type animals and a maximal lifespan of approximately 100 days, but also significantly increased resistance to oxidative and heat stress. Synergistic lifespan extension depends on the transcription factor DAF-16 (FOXO). mRNA-seq experiments revealed that the mRNA levels of DAF-16 Class I genes, which are activated by DAF-16, were further elevated in the DAF-2;set double mutants. Among these genes, tts-1, F35E8.7, ins-35, nhr-62, sod-3, asm-2, and Y39G8B.7 are required for the lifespan extension of the DAF-2;set-21 double mutant. In addition, treating DAF-2 animals with the H3K9me1/2 methyltransferase G9a inhibitor also extends lifespan and increases stress resistance. Therefore, investigation of DAF-2 and H3K9me1/2 deficiency-mediated synergistic longevity will contribute to a better understanding of the molecular mechanisms of aging and therapeutic applications.