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一氧化氮检测试剂盒 (DAF-FM DA, 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
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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
一氧化氮检测试剂盒（DAF‑FM DA）以 DAF‑FM DA 作为荧光探针，可快速、高灵敏地检测培养细胞内一氧化氮的含量变化。
DAF‑FM DA，全称 4‑Amino‑5‑Methylamino‑2,7‑Difluorofluorescein Diacetate，亦称为 DAF‑FM Diacetate，是用于一氧化氮（nitric oxide, NO）定量检测的荧光探针。相较于传统一氧化氮荧光探针 DAF‑2 DA，其主要优势如下：
①DAF‑FM DA 与 NO 反应生成的荧光产物在 pH 5.5 以上环境中不受 pH 干扰；
②反应产物荧光稳定性更强，不易发生荧光淬灭，更利于样品检测；
③DAF‑FM DA 最低检出浓度可达 3 nM，检测灵敏度较 DAF‑2 DA 提升近 2 倍。
检测原理：DAF‑FM DA 能够穿透活细胞膜，进入细胞后经胞浆酯酶水解生成细胞膜非通透性的 DAF‑FM。DAF‑FM 本身荧光极弱，与 NO 发生反应后生成荧光素‑苯骈三氮唑（benzotriazole），产生强烈的绿色荧光。
本试剂盒提供的 DAF‑FM DA 为 5 mM 储存液。工作终浓度建议为 1‑10 μM，推荐最优终浓度 5 μM；外源阳性刺激物 NOup（200×）推荐终浓度 0.5‑2 ×，推荐最优终浓度 1 ×。本试剂盒兼容荧光显微镜、激光共聚焦显微镜、流式细胞仪、荧光酶标仪等主流荧光检测设备；试剂盒配套提供检测缓冲液及阳性对照 NOup，实验操作简便。
本试剂盒适用于细胞内一氧化氮水平检测，支持实时动态检测。若收集细胞后再进行探针装载：小包装至少可检测 40 个样品，中包装至少可检测 200 个样品。按孔板染色体系计算：6 孔板单孔体系 1 mL，小包装约可检测 20 次，中包装约可检测 100 次；48 孔板单孔体系 200 μL，小包装约可检测 100 次，中包装约可检测 500 次；96 孔板单孔体系 100 μL，小包装约可检测 200 次，中包装约可检测 1000 次。
产品组成
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使用方法
（一）阳性对照的设置
1.在设置为阳性对照的孔中提前 30 min 加入 NOup（200×），使用完全培养基稀释即可。
2.吸出培养基，在离心管中用检测缓冲液将 DAF-FM DA 稀释 1000 倍，同时再加入 1/200 的 NOup，混合均匀后，加入孔中。37 ℃孵育 20 min。
3.PBS 洗涤 2 - 3 次后，显微镜下观察拍照。
（二）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，用本试剂盒提供的检测缓冲液稀释 DAF-FM DA，使终浓度为 5 μM。去除细胞培养液，PBS 洗涤 2 次后，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 min。用 PBS 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，用本试剂盒提供的检测缓冲液稀释 DAF-FM DA，使终浓度为 5 μM。离心收集细胞，PBS 洗涤 2 次后，用稀释好的 DAF-FM DA 重悬细胞，细胞浓度为 1 × 10⁶～2 × 10⁷ 个 /mL，37 ℃细胞培养箱内孵育 20 min。上述操作可以在离心管内进行。每隔 3 - 5 min 颠倒混匀一下，使探针和细胞充分接触。用 PBS 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。
（三）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。
（四）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。
（五）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 min 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 2-8℃、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、仅在阳性对照孔中加入 NOup 作为阳性对照，其余孔不必加入 NOup。
5、为了您的安全和健康，请穿实验服并戴一次性手套操作。
Nitric Oxide Assay Kit (DAF-FM DA, Green) uses DAF‑FM DA as fluorescent probe to rapidly and sensitively detect changes in intracellular nitric oxide levels in cultured cells.
DAF‑FM DA, full name 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). Compared with the conventional NO fluorescent probe DAF‑2 DA, it exhibits the following major advantages:
① The fluorescent product formed by reaction between DAF‑FM DA and NO is pH‑insensitive above pH 5.5;
② The reaction product possesses higher fluorescence resistance to quenching, which facilitates detection;
③ DAF‑FM DA achieves a limit of detection as low as 3 nM, representing nearly 2‑fold improvement in sensitivity relative to DAF‑2 DA.
Principle of Detection: DAF‑FM DA readily crosses the plasma membrane of live cells. Once inside cells, it is hydrolyzed by cytosolic esterases into membrane‑impermeable DAF‑FM. DAF‑FM shows very weak intrinsic fluorescence; upon reaction with NO, it forms fluorescein‑benzotriazole and generates intense green fluorescence.
DAF‑FM DA supplied in this kit is a 5 mM stock solution. The recommended working final concentration ranges from 1‑10 μM, with an optimal concentration of 5 μM. For exogenous stimulant NOup (200×), the suggested final concentration is 0.5‑2 ×, and 1 × is optimal. This kit is compatible with mainstream fluorescence‑detecting instruments including fluorescence microscope, laser scanning confocal microscope (LSCM), flow cytometer and fluorescence microplate reader. Assay buffer and positive control NOup are included for convenient operation.
This kit is suitable for determining intracellular NO levels and supports real‑time detection. When probe loading is performed after cell harvest: the small‑size kit enables no less than 40 assays, and the medium‑size kit no less than 200 assays. For well‑plate staining: at 1 mL per well in 6‑well plates, approximately 20 assays for small size and 100 assays for medium size; at 200 μL per well in 48‑well plates, approximately 100 assays for small size and 500 assays for medium size; at 100 μL per well in 96‑well plates, approximately 200 assays for small size and 1000 assays for medium size.
Product Components
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Protocol
(I) Preparation of Positive Control
1.Add NOup (200×) to designated positive control wells 30 min in advance, diluted with complete culture medium.
2.Aspirate the culture medium. Dilute DAF-FM DA 1000-fold with Assay Buffer in a centrifuge tube, then add NOup to a final dilution of 1/200. Mix thoroughly and add the mixture to the wells. Incubate at 37 ℃ for 20 min.
3.Wash 2 - 3 times with PBS, then observe and capture images under a microscope.
(II) 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 1:1000 using the Assay Buffer provided in the kit to a final concentration of 5 μM. Remove culture medium and wash cells twice with PBS, then 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 min. Wash cells three times with PBS to fully remove extracellular unloaded DAF-FM DA.
Probe Loading after Cell Harvest: Dilute DAF-FM DA 1:1000 using the Assay Buffer provided in the kit to a final concentration of 5 μM. Harvest cells by centrifugation and wash twice with PBS. Resuspend the cell pellet in diluted DAF-FM DA at a cell density of 1 × 10⁶ – 2 × 10⁷ cells/mL, and incubate in a 37 ℃ cell incubator for 20 min. All operations can be performed in centrifuge tubes. Invert the tube gently every 3 - 5 min to ensure sufficient contact between the probe and cells. Wash cells three times with PBS 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.
(III) 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.
(IV) 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.
(V) 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 min according to experimental conditions.
Precautions
1.BSA and phenol red interfere with detection using this fluorescent probe and should be avoided.
2.Aliquot the kit upon first use and store at -20 ℃ to prevent repeated freeze-thaw cycles. Prepare the fluorescent probe working solution immediately before use; long-term storage is not permitted.
3.DAF-FM DA may solidify and adhere to the bottom, wall or cap of centrifuge tubes under low temperature conditions such as 2 - 8 ℃ or ice bath. Warm the tube at 20 - 25 ℃ briefly until fully thawed before use.
4.Add NOup only to positive control wells; do not add NOup to other wells.
5.Wear a lab coat and disposable gloves for personal safety and protection.
规格
别名
DAF-FM DA 一氧化氮荧光检测试剂盒,NO 荧光染色试剂盒（DAF-FM DA）
英文别名
DAF-FM DA Nitric Oxide Fluorescence Assay Kit
规格或纯度
BioReagent, 即用型, 适用于荧光分析, 无菌
英文名称
Nitric Oxide Assay Kit (DAF-FM DA, Green)
应用
细胞代谢
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,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)
495nm
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 | 500T | 储存 |
| N1527960A | DAF-FM DA（5mM） | 液体 | 20μL | 100 μL | -20℃. 避光 |
| N1527960B | 检测缓冲液 | 液体 | 50mL | 250 mL | -20℃. 避光 |
| N1527960C | NOup（200×） | 液体 | 20μL | 100 μL | -20℃. 避光 |
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