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DAF-FMDA (一氧化氮荧光探针, 绿色)
有货
为什么选择此级别
BioReagent,即用型,无菌,适用于荧光分析 级 ，适用于对基线干扰要求严格的色谱和分析工作流程。
储存与运输
避光,-20°C储存。超低温运输 。请查阅批次 COA 获取详细规格。
质量文档
SDS、COA、产品数据表及规格说明书均可下载。可通过批号查询获取批次 COA。
文献证明
在色谱分析、有机合成和交叉偶联反应领域已被 0 篇同行评审文献引用。
概述
是否无菌：是
DAF‑FM DA（4‑Amino‑5‑Methylamino‑2,7‑Difluorofluorescein Diacetate，亦称 DAF‑FM Diacetate）是一种用于一氧化氮（NO）定量检测的荧光探针。
检测原理：DAF‑FM DA 可自由穿透活细胞膜，进入细胞后，被胞浆内酯酶催化水解，生成不能穿膜的极性产物 DAF‑FM。DAF‑FM 本身仅呈现微弱荧光，但在与 NO 反应后，迅速转化为荧光素‑苯骈三氮唑（benzotriazole）衍生物，释放出强烈绿色荧光。产物可用荧光显微镜、激光共聚焦显微镜、流式细胞仪、荧光分光光度计或荧光酶标仪等多种平台检测。
核心优势
与早期 NO 荧光探针 DAF‑2 DA 相比，DAF‑FM DA 实现了三大技术升级：
• pH 不敏感：反应产物在 pH ≥ 5.5 范围内荧光强度保持恒定，适配更广泛的实验体系。
• 光稳定性优异：荧光产物不易淬灭，信号持久，为成像与流式分析提供更充裕的检测窗口。
• 灵敏度显著提升：最低检测浓度可达 3 nM，较 DAF‑2 DA 提高近 2 倍，满足高灵敏度 NO 检测需求。
产品形式：即用型 DMSO 溶液，淡黄色，浓度为 5 mM。本规格通常可满足 不少于 100 个样品 的检测需求。
产品组成
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使用说明
1.装载探针
对于刺激时间较短（通常为 2 小时以内）的细胞，先装载探针，后用适当的阳性对照及待测药物刺激细胞。对于细胞刺激时间较长（通常为 6 小时以上）的细胞，先用适当的阳性对照及待测药物刺激细胞，后装载探针。
原位装载探针：本方法仅适用于贴壁培养细胞。按照 1:1000 比例，用本试剂盒提供的 DAF-FM DA 稀释液稀释 DAF-FM DA，使终浓度为 5 μM。去除细胞培养液，加入适当体积稀释好的 DAF-FM DA。加入的体积以能充分盖住细胞为宜，通常对于六孔板的一个孔加入稀释好的 DAF-FM DA 的体积为 1 mL。37 ℃细胞培养箱内孵育 20 分钟。用 PBS (pH 7.4) 洗涤细胞三次，以充分去除未进入细胞内的 DAF-FM DA。
收集细胞后装载探针：按照 1:1000 比例，用本试剂盒提供的 DAF-FM DA 稀释液稀释 DAF-FM DA，使终浓度为 5 μM。细胞收集后，用稀释好的 DAF-FM DA 重悬细胞，细胞浓度为 1 × 10⁶ – 2 × 10⁷ cell/mL，37 ℃细胞培养箱内孵育 20 分钟。上述操作可以在离心管内进行。每隔 3 – 5 分钟颠倒混匀一下，使探针和细胞充分接触。用 PBS (pH 7.4) 洗涤细胞三次，以充分去除未进入细胞内的 DAF-FM DA。直接用适当的阳性对照或待测药物刺激细胞，或把细胞等分成若干份后再刺激细胞。
2.检测
对于原位装载探针的样品可以用激光共聚焦显微镜直接观察（普通荧光显微镜观察效果相对较差），或收集细胞后用荧光分光光度计、荧光酶标仪或流式细胞仪检测。对于收集细胞后装载探针的样品可以用荧光分光光度计、荧光酶标仪或流式细胞仪检测，也可使用激光共聚焦显微镜直接观察。
3.参数设置
使用 495 nm 激发波长，515 nm 发射波长，实时、逐时间点或单时间点检测刺激前后荧光的强弱。DAF-FM 和一氧化氮反应产物的荧光光谱和 fluorescein 非常相似，可以采用检测 fluorescein 的参数设置进行检测，也可采用检测 FITC 的参数设置。
4.其它说明
上述推荐的 DAF-FM DA 的工作浓度为 5 μM，对于某些细胞，如果未刺激的阴性对照细胞荧光较强，可以按照 1:2000 – 1:5000 的比例稀释 DAF-FM DA，使装载探针时 DAF-FM DA 的浓度为 1 – 2.5 μM。相反，如果待测药物刺激后荧光较弱，可以把 DAF-FM DA 的工作浓度调整为 10 μM，以提高检测的灵敏度。另外，探针装载的时间也可以根据情况在 15 – 60 分钟内适当进行调整。
注意事项
1、BSA 和酚红对该荧光探针的检测有干扰，需避免。
2、第一次使用前请将本品分装并于-20℃冻存，避免反复冻存；荧光探针工作液需立刻使用，不能长时间保存。
3、DAF-FM DA 在 4℃、冰浴等较低温度情况下会凝固而粘在离心管管底、管壁或管盖内，可以 20-25℃温浴片刻至全部融解后使用。
4、为了您的安全和健康，请穿实验服并戴一次性手套操作。
DAF‑FM DA（4‑Amino‑5‑Methylamino‑2,7‑Difluorofluorescein Diacetate，also known as DAF‑FM Diacetate）is a fluorescent probe for quantitative detection of nitric oxide (NO).
Detection Principle: DAF‑FM DA readily crosses the plasma membrane of living cells. Once inside the cell, it is hydrolyzed by cytosolic esterases to yield DAF‑FM, a polar product incapable of membrane permeation. DAF‑FM itself exhibits only faint fluorescence. Upon reaction with NO, it is rapidly converted into a fluorescein‑benzotriazole derivative that emits intense green fluorescence. The resultant product can be assayed on multiple platforms, including fluorescence microscopes, laser scanning confocal microscopes, flow cytometers, fluorescence spectrophotometers, or fluorescence microplate readers.
Key Advantages
Compared with the earlier NO fluorescent probe DAF‑2 DA, DAF‑FM DA features three major technical improvements:
• pH‑insensitive: Fluorescence intensity of the reaction product remains constant at pH ≥ 5.5, compatible with a broader range of experimental systems.
• Excellent photostability: The fluorescent product is resistant to photobleaching and generates sustained signals, providing an extended detection window for imaging and flow cytometric analysis.
• Markedly enhanced sensitivity: It achieves a limit of detection as low as 3 nM, nearly two‑fold higher sensitivity than DAF‑2 DA, meeting the requirements for high‑sensitivity NO measurement.
Product Form: Ready‑to‑use pale‑yellow DMSO solution at a concentration of 5 mM. This format is generally sufficient for no less than 100 samples.
Product Components
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Instructions for Use
1.Probe Loading
For cells with a short stimulation duration (generally within 2 hours), load the probe first, then treat cells with appropriate positive control and test compounds. For cells requiring long-term stimulation (generally more than 6 hours), treat cells with appropriate positive control and test compounds first, then load the probe.
In-situ Probe Loading: This method is only applicable to adherent cells. Dilute DAF-FM DA at a ratio of 1:1000 with the DAF-FM DA diluent provided in the kit to achieve a final concentration of 5 μM. Remove the culture medium and add an appropriate volume of diluted DAF-FM DA sufficient to cover the cell monolayer. Typically, 1 mL of diluted DAF-FM DA is used per well of a 6-well plate. Incubate in a 37 ℃ cell incubator for 20 minutes. Wash cells three times with PBS (pH 7.4) to fully remove extracellular unloaded DAF-FM DA.
Probe Loading after Cell Harvest: Dilute DAF-FM DA at a ratio of 1:1000 with the DAF-FM DA diluent provided in the kit to achieve a final concentration of 5 μM. After cell collection, resuspend the cell pellet with diluted DAF-FM DA at a cell density of 1 × 10⁶ – 2 × 10⁷ cell/mL, and incubate in a 37 ℃ cell incubator for 20 minutes. All operations can be performed in centrifuge tubes. Invert the tube gently every 3 – 5 minutes to ensure sufficient contact between the probe and cells. Wash cells three times with PBS (pH 7.4) to fully remove extracellular unloaded DAF-FM DA. Treat cells directly with appropriate positive control or test compounds, or aliquot the cell suspension before treatment.
2.Detection
Samples prepared by in-situ probe loading can be visualized directly under a laser scanning confocal microscope (ordinary fluorescence microscopes yield relatively poor results), or harvested for detection using a fluorescence spectrophotometer, fluorescence microplate reader or flow cytometer. Samples prepared by probe loading after cell harvest can be analyzed with a fluorescence spectrophotometer, fluorescence microplate reader or flow cytometer, and can also be observed directly under a laser scanning confocal microscope.
3.Instrument Parameter Settings
Use an excitation wavelength of 495 nm and an emission wavelength of 515 nm to detect fluorescence intensity before and after stimulation in real time, at sequential time points or a single time point. The fluorescence spectrum of the reaction product of DAF-FM and nitric oxide is highly similar to that of fluorescein. Detection parameters for fluorescein or FITC can be adopted.
4.Additional Notes
The recommended working concentration of DAF-FM DA is 5 μM. For certain cell types with high background fluorescence in unstimulated negative control cells, DAF-FM DA can be diluted at 1:2000 – 1:5000 to achieve a loading concentration of 1 – 2.5 μM. Conversely, if fluorescence signal is weak after treatment with test compounds, the working concentration of DAF-FM DA can be adjusted to 10 μM to improve detection sensitivity. The probe incubation time can also be optimized within the range of 15 – 60 minutes according to experimental conditions.
Precautions
1.BSA and phenol red interfere with the detection of this fluorescent probe and should be avoided.
2.Aliquot the product before first use and store at -20 ℃ to prevent repeated freeze-thaw cycles. The fluorescent probe working solution must be prepared immediately before use and cannot be stored for a long time.
3.DAF-FM DA tends to solidify and adhere to the bottom, wall or cap of centrifuge tubes under low-temperature conditions such as 4 ℃ or ice bath. Warm the tube at 20 - 25 ℃ briefly until fully thawed prior to use.
4.Wear a lab coat and disposable gloves for personal safety during operation.
规格
规格或纯度
BioReagent, 即用型, 适用于荧光分析, 无菌, 5mM in DMSO
英文名称
DAF-FM DA (Nitric Oxide Fluorescent Probe, Green)
应用
Metabolic Assay
样品类型
细胞
检测方法
荧光法
定量&半定量
半定量
检测仪器
流式细胞仪,荧光显微镜,荧光酶标仪,荧光分光光度计,激光共聚焦显微镜
检测波长
Ex/Em:495/515nm
敏感性
Light-sensitive
Emission(nm)
515nm
Excitation(nm)
Blue Laser(488nm)
Ex/Em(nm)
Ex:495nm;Em:515nm
储存与运输
储存条件
避光,-20°C储存
运输条件
超低温运输
稳定性与储存
Store at -20℃ long term (12 months). Store in the dark.
组分与储存
| 组分货号 | 组分名称 | 物理外观 | 100T | 储存 |
| D1527918A | DAF-FM DA | 液体 | 20μL | -20℃，避光 |
| D1527918B | DAF-FM DA稀释液 | 液体 | 50mL | -20℃，避光 |
技术文档
高级数据
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