Hydroxyphenyl fluorescein (HPF) 羟苯基荧光素

描述

Hydroxyphenyl fluorescein (HPF) 羟苯基荧光素

 

产品信息

 

产品描述

氨基苯基荧光素（APF）和羟苯基荧光素（HPF）是由T. Nagano开发的两种活性氧（Reactive oxygen species, ROS）指示剂，比H2DCFDA（DCFH-DA, DCFH）具更优秀的特异性和稳定性。APF和HPF显示有限的非选择性反应，和相对较高的光诱导氧化抗性。APF和HPF具细胞膜渗透性，本身无荧光，直到与羟基自由基或过氧亚硝基阴离子反应，还能与次氯酸阴离子反应，产生强绿色荧光（Ex/Em，~490/515nm），可用荧光显微镜、高通量成像仪、荧光酶标板或流式细胞仪检测。

 

本品以粉末形式提供，可直接溶于DMF配制成5mM储存液，常用工作浓度范围1-10μM，需根据自身的实验体系或参考文献进行优化。

 

产品特性

1）CAS NO.：359010-69-8

2）同义名：3′-p-(hydroxyphenyl) fluorescein (HPF)羟苯基荧光素

3）分子式：C26H16O6

4）分子量：424.4

5）纯度：≥95%

6）Ex/Em：492/515nm

7）溶解性：溶于DMSO（10mM）、DMF（5mM）

8）化学结构：

保存与运输方法

保存：-20ºC避光干燥保存，至少一年有效。

运输：冰袋运输。 

使用方法

一、储存液制备

将低温保存的1mg粉末置于室温回温至少20min，之后加入471μl 无水DMF配置成5mM储存液。充分溶解和混匀后，按照单次用量分装，-20ºC避光干燥保存，避免反复冻融。

【注】：HPF能溶于DMSO（10mM），但有数据显示DMSO是羟基自由基的淬灭剂，会干扰检测和降低灵敏性。因此，建议用DMF作为溶剂。

二、溶液体系检测

正式实验前，取一管HPF储存液置于室温回温，使其充分融解。之后用合适的缓冲液（比如HHBS缓冲液，PBS缓冲液）稀释到所需的工作浓度。建议起始浓度为1-10μM，需根据自身的实验体系或参考文献进行优化。

三、细胞体系检测

以下实验步骤仅做参考，最佳的工作浓度、孵育时间和温度需根据实际情况进行调整。在加入HPF工作液之前按需处理细胞。

1）   根据具体用量，将DMF储存液用HHBS缓冲液或其他生理缓冲液稀释成1-10μM工作液。

2）   根据实验设计处理细胞（比如，RASM细胞用50-100nM 血管紧张素II处理3-5h）。

3）   取步骤1）准备的1-10μM工作液加入细胞，37ºC孵育20-60min。

4）   吸掉染色工作液，用HHBS或其他生理缓冲液替换。

5）   用合适的荧光仪器（Ex/Em=492/515nm）来检测。

【注1】：BSA和酚红会影响荧光，需谨慎使用。

【注2】：HPF和APF能用于溶液体系或胞内ROS检测。

注意事项

1）   APF和HPF仅用于科研研究，不可用于体外诊断研究。

2）   APF和HPF产生的荧光相对于其他活性氧探针比如DCFH弱很多，这是探针本身的特性决定的（见附表1 活性氧探针（HPF, APF, DCFH）对各种活性氧物质的反应特异性）。

3）   为了您的安全和健康，请穿实验服并戴一次性手套操作。

相关产品

货号	名称	规格
MX4801-1KIT	Reactive Oxygen Species (ROS) Assay Kit 活 性氧（ROS）检测试剂盒	100~500T
MX4802-50MG	H2DCFDA (DCFH-DA, DCFH) 活性氧（ROS）荧光探针	50mg
MX4804-1MG	Aminophenyl fluorescein (APF) 氨基苯基荧光素	1mg
MX4805-1MG	Hydroxyphenyl fluorescein (HPF) 羟苯基荧光素	1mg
MX4455-1MG	Dihydrorhodamine 123 (DHR 123) 二氢罗丹明123	1mg
MM0706-1G	Luminol 鲁米诺	1g
MX4606-500UG	Coelenterazine Native 天然腔肠素	500μg

附表1 活性氧探针（HPF, APF, DCFH）对各种活性氧物质的反应特异性

ROS	ROS产生方法	HPF (MX4805)	APF (MX4804)	DCFH (MX4802)
HO•	100 µM高氯酸亚铁(II)+1 mMH2O2	730	1200	7400
ONOO–	3 µM (终浓度) ONOO–	120	560	6600
OCl-	3 µM (终浓度) OCl-	6	3600	86
1O2	100 µM 3-(1,4-dihydro-1,4-epidioxy-1-naphthyl) propionic acid	5	9	26
•O2–	100 µM KO2	8	6	67
H2O2	100 µM of H2O2	2	＜1	190
NO	100 µM NOC-7	6	＜1	150
ROO•	100 µM AAPH	17	2	710
Autoxidation	荧光光照2.5h	＜1	＜1	2000

 

 

 — —Written/Edited by V. Shallan【版权归集奇生物/MKBio懋康所有】

文献引用：

[1]Zhen WY et al. Reductive Surfactant-Assisted One-Step Fabrication of BiOI/BiOIO3 Heterojunction Biophotocatalyst for Enhanced Photodynamic Theranostics Overcoming Tumor Hypoxia. Nanoscale Horiz., 2019, 4, 720-726

Hydroxyphenyl fluorescein (HPF) was purchased from Shanghai Maokang Bio. Co. (Shanghai, China). HPF was used to detect the generation of •OH. Typically, 1 mL BB NCs aqueous solution (80 µg mL -1 ) containing with HPF (1 µM) were irradiated by 650 nm laser (0.5 W cm-2 ), the fluorescence of HPF was detected after 15 min. HPF aqueous solution with laser served as control.

  

[2]Liu Yang et al. Defect modified zinc oxide with augmenting sonodynamic reactive oxygen species generation. Biomaterials. Volume 251, August 2020, 120075

Hydroxyphenyl fluorescein (HPF) was purchased from Shanghai Maokang Bio. Co. (Shanghai, China).

[3]Xu X, Huang B, Zeng Z, Chen J, Huang Z, Guan Z, Chen M, Huang Y, Zhao C. Broaden sources and reduce expenditure: Tumor-specific transformable oxidative stress nanoamplifier enabling economized photodynamic therapy for reinforced oxidation therapy. Theranostics 2020; 10(23):10513-10530. doi:10.7150/thno.49731.

Hydroxyphenyl fluorescein (HPF) was purchased from Shanghai Maokang Bio. Co. (Shanghai, China). For detecting intracellular •OH generation, treated B16F10 cells were rinsed and stained with specific •OH probe HPF (10 μM in PBS) for 60 min, followed by rinsed with PBS and imaged using CLSM. To further confirm the Fe(II) consumption, after treatment with H2O2 (positive control), CA and HA@CA for 12 h, the cells were stained with a ferric ion probe, 3′,6′-bis (diethylamino)-2-(4-oxopent-2-en-2-ylamino) spiro (isoindoline-1,9′-xanthen)-3-one (10 μM) at 37 °C for 30 min. Finally, the treated cells were rinsed with PBS and observed by CLSM.

[4]Tang H, Li C, Zhang Y, et al. Targeted Manganese doped silica nano GSH-cleaner for treatment of Liver Cancer by destroying the intracellular redox homeostasis. Theranostics. 2020;10(21):9865-9887. Published 2020 Aug 2. doi:10.7150/thno.46771

Hydroxyphenyl fluorescein (HPF) was supplied by Shanghai Mao Kang biotechnology Co., Ltd. For evaluating the different types of ROS, DHE and HPF were selected as probes to assess the generation of intracellular •O2- and •OH, respectively. Briefly, HepG2 cells (5×105 cells per well) were seeded in a 6-well plate and incubated overnight. After that, Free SFB, MnMSN@SFB or FaPEG-MnMSN@SFB at different concentrations (10, 20, and 40 µg/mL of SFB equivalent) were added and treated for 6 h. Cells were then stained with DHE (20 µM) for another 45 min. Eventually, fluorescence microscope was employed to observe the fluorescence of cells at the wavelength of 535 nm. Moreover, to assess the generation of intracellular •OH, HepG2 cells (1×105 cells per well) were seeded into confocal dishes and incubated for 12 h. Then, Free SFB, MnMSN@SFB or FaPEG-MnMSN@SFB at different concentrations (10, 20, and 40 μg/mL of SFB equivalent) were added, meanwhile HPF (10 µM) was added to capture generative intracellular •OH, following by co-incubation for 6 h. Eventually, CLSM was used to observe the fluorescence of cells at 490 nm.

【5】

Lili Zhang, Jiabao Huang, Shiqi Su, Xiaochun Wei, Lin Yang, Huanhuan Zhao, Jianqiang Yu, Jie Wang, Jiyun Hui, Shiya Hao, Shanshan Song, Yanyan Cao, Maoshuai Wang, Xiaowei Zhang, Yanyan Zhao, Zhiyong Wang, Weiqing Zeng, Hen-Ming Wu, Yuxiang Yuan, Xiansheng Zhang, Alice Y. Cheung, Qiaohong Duan,

FERONIA receptor kinase-regulated reactive oxygen species mediate self-incompatibility in Brassica rapa, Current Biology, 2021, ISSN 0960-9822, https://doi.org/10.1016/j.cub.2021.04.060.

Probe 20,70-Dichlorodihydrofluorescein diacetate (H2DCFDA), dihydroethidium (DHE), hydroxyphenyl fluorescein (HPF) are commonly used for ROS detection. HPF was used at 10 mM in 0.2 M PBS buffer (pH 6.1) for 2 hours. Comparable results were obtained in experiments tested with all three dyes, providing confidence for the observed changes in ROS levels.

【6】

Mengsi Wu, Zhiyong Liu, Weian Zhang. An ultra-stable bio-inspired bacteriochlorin analogue for hypoxia-tolerant photodynamic therapy. Chemical Science 2021, 12 (4) , 1295-1301. https://doi.org/10.1039/D0SC05525E

Singlet oxygen sensor green (SOSG), dihydroethidium (DHE), 3’-(4-hydroxyphenyl) fluorescein (HPF), Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM), Hoechst 33342 and 3-(4,5- dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Shanghai Maokang Biotechnology Co.,Ltd.  Hydroxyl radical (•OH) was detected by fluorescence spectroscopy using hydroxyphenyl fluorescein (HPF), which reacts with •OH to produce high fluorescence emission at 530 nm. HPF was dissolved in DMF to gain 5 mM HPF solution. Then, 20 μL HPF was added into the solution of 2 mL FBC or FBC nanogels. The fluorescence changes were detected after illumination with 750 nm laser, and the excitation wavelength was 490 nm.

【7】

Jinghua An, Xiaoting Song, Wenbo Wan, Yanzheng Chen, Haibin Si, Huichuan Duan, Lu Li*, and Bo Taing*. Kinetics of the Photoelectron-Transfer Process Characterized by Real-Time Single-Molecule Fluorescence Imaging on Individual Photocatalyst Particles. ACS Catal. 2021, 11, 12, 6872–6882 Publication Date:May 27, 2021 https://doi.org/10.1021/acscatal.1c00983

HPF and Amplex red from maokang biotechnology co. ltd.