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。

【注】：与DMSO相比，DMF的细胞毒性相对较高，建议用户在使用过程中，在达到理想荧光信号的前提下使用最低浓度的探针。如果待检细胞在培养的过程中就发现比较敏感（或不好培养），建议在使用HPF之前，先做一组溶剂对照组来观察细胞对DMF的敏感程度。

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

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

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

上海懋康生物科技有限公司是一家涉足于生命科学和生物技术领域研究的试剂、仪器和实验室消耗品与实验服务工作，主要从事细胞生物学、植物学、分子生物学、免疫学、生物化学、蛋白组学。生物制药与诊断试剂研发生产等领域。 本公司秉承“以人为本，以诚为信、合同守信”的经营理念。坚持"品质保障"的原则为广大客户提供优质产品。

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] Zhang L, Huang J, Su S, Wei X, Yang L, Zhao H, Yu J, Wang J, Hui J, Hao S, Song S, Cao Y, Wang M, Zhang X, Zhao Y, Wang Z, Zeng W, Wu HM, Yuan Y, Zhang X, Cheung AY, Duan Q. FERONIA receptor kinase-regulated reactive oxygen species mediate self-incompatibility in Brassica rapa. Curr Biol. 2021 Jul 26;31(14):3004-3016.e4. doi: 10.1016/j.cub.2021.04.060. Epub 2021 May 19. PMID: 34015250.

HPF Maokang Bio-Technology

FM4-64 Maokang Bio-Technology

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.

[8] Wen Li et al. Mutual-reinforcing sonodynamic therapy against Rheumatoid Arthritis based on sparfloxacin sonosensitizer doped concave-cubic rhodium nanozyme. Biomaterials. Volume 276, September 2021, 121063

Hydroxyphenyl fluorescein (HPF) probe and ROS Green™ H 2 O 2 probe were purchased from Shanghai Maokang Biotechnology Co., Ltd

[9] Qiu-Lin Yan, Xin-Ye Wang, Ming Bai, Xin Zhang, Shao-Jiang Song, Guo-Dong Yao,

Sesquiterpene lactones from Elephantopus scaber exhibit cytotoxic effects on glioma cells by targeting GSTP1,

Bioorganic Chemistry,Volume 129,2022,106183,ISSN 0045-2068,https://doi.org/10.1016/j.bioorg.2022.106183.

To evaluate the different types of ROS, DHE, commercial ROSGreen H 2 O 2 probe, HPF and C11-BODIPY 581/591(Maokang Biotechnology, Shanghai, China) were selected as probes to assess the intracellular superoxide, the intracellular H 2 O 2, the intracellular ⋅OH generation and the intracellular lipid oxidation, respectively.

[10] Yang RQ, Wang PY, Lou KL, Dang YY, Tian HN, Li Y, Gao YY, Huang WH, Zhang YQ, Liu XL, Zhang GJ. Biodegradable Nanoprobe for NIR-II Fluorescence Image-Guided Surgery and Enhanced Breast Cancer Radiotherapy Efficacy. Adv Sci (Weinh). 2022 Apr;9(12):e2104728. doi: 10.1002/advs.202104728. Epub 2022 Feb 16. PMID: 35170876; PMCID: PMC9036023.

Moreover, to detect intracellular hydroxyl radical generation, R&HV-Gd@ICG + X ray-treated 4T1 cells were rinsed and stained with a specific •OH probe (hydroxyphenyl fluorescein; HPF; Shanghai Maokang Bio., Co.; 10 × 10⁻⁶ m dissolved in phosphate-buffered saline (PBS)) for 30 min, rinsed with PBS, and imaged using a confocal microscope.

[11] Wu M, Chen C, Liu Z, Tian J, Zhang W. Regulating the bacterial oxygen microenvironment via a perfluorocarbon-conjugated bacteriochlorin for enhanced photodynamic antibacterial efficacy. Acta Biomater. 2022 Apr 1;142:242-252. doi: 10.1016/j.actbio.2022.02.013. Epub 2022 Feb 17. PMID: 35183779.

Singlet oxygen sensor green (SOSG), dihydroethidium (DHE), and 3’-(4-hydroxyphenyl) fluorescein (HPF) were purchased from Shanghai Maokang Biotechnology

[12] Chao S, Shen Z, Pei Y, Lv Y, Chen X, Ren J, Yang K, Pei Z. Pillar[5]arene-based supramolecular photosensitizer for enhanced hypoxic-tumor therapeutic effectiveness. Chem Commun (Camb). 2021 Aug 3;57(62):7625-7628. doi: 10.1039/d1cc02959b. PMID: 34235523.

Dihydrorhodamine 123 (DHR123), Dihydroethidium (DHE), hydroxyphenyl fluorescein (HPF), singlet oxygen sensor green (SOSG) were purchased from MKbio (China).

[13] Li, Z.; Liu, S.; Zhu, T.; An, X.; Wei, X.; Zhang, J.; Wu, S.; Dong, Z.; Long, Y.; Wan, X. The Loss-Function of the Male Sterile Gene ZmMs33/ZmGPAT6 Results in Severely Oxidative Stress and Metabolic Disorder in Maize Anthers. Cells 2022, 11, 2318. https://doi.org/10.3390/cells11152318

For ROS measurement, H₂DCF-DA (D6883, Sigma-Aldrich, USA) was dissolved in DMSO (D2650, Sigma-Aldrich, USA) to prepare a 10 mM stock solution. Then, it was diluted to 5 μM as a working solution in 1× HHBS Buffer (MS3510, Maokang Bio-Technology, Shanghai, China). For •OH measurement, HPF (MX4805, Maokang Bio-Technology, China) was dissolved in DMF (227056, Sigma-Aldrich, USA) to prepare a 5 mM stock solution. 

[14] Zhang YY, Ren H, Yan QL, Li YL, Liu Q, Yao GD, Song SJ. SCP-7, a germacrane-type sesquiterpene lactone derivative, induces ROS-mediated apoptosis in NSCLC cells in vitro and in vivo. Eur J Pharmacol. 2022 Jun 15;925:174989. doi: 10.1016/j.ejphar.2022.174989. Epub 2022 Apr 28. PMID: 35490722.

 HPF (Hydroxyphenyl fluorescein) and ROS GreenTM H2 O 2 Probe was purchased from Shanghai Maokang Bio. Co. (Shanghai, China). 

[15] Liao K, Niu B, Dong H, He L, Zhou Y, Sun Y, Yang D, Wu C, Pan X, Quan G. A spark to the powder keg: Microneedle-based antitumor nanomedicine targeting reactive oxygen species accumulation for chemodynamic/photothermal/chemotherapy. J Colloid Interface Sci. 2022 Dec 15;628(Pt B):189-203. doi: 10.1016/j.jcis.2022.08.042. Epub 2022 Aug 11. PMID: 35994900.

Hydroxyphenyl fluorescein (HPF, 99 %) was purchased from Shanghai Maokang Biotechnology Co., ltd. (Shanghai, China).

[16]Bortezomib prodrug catalytic nanoreactor for chemo/chemodynamic therapy and macrophage re-education

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

[17] Cai J, Liu LF, Qin Z, et al. Natural Morin-Based Metal Organic Framework Nanoenzymes Modulate Articular Cavity Microenvironment to Alleviate Osteoarthritis. Research (Washington, D.C.). 2023 ;6:0068. DOI: 10.34133/research.0068. PMID: 36930778; PMCID: PMC10013961.

Subsequently, the chondrocytes were respectively cocultured with 20 μM DCF (Solarbio, China) for total ROS testing, 10 μM HPF (maokangbio, China) for ·OH testing, or 5 μM DHE (Beyotime Biotechnology, China) for ·O₂⁻ testing before PBS washing 3 times. Finally, the fluorescent intensity of chondrocytes was observed by fluorescent microscopy and the corresponding quantification was analyzed by ImageJ.

[18] Vibsane-type diterpenoids from Viburnum odoratissimum inhibit hepatocellular carcinoma cells via the PI3K/AKT pathway

ROS Green TM H 2 O 2 probe, Hydroxyphenyl fluorescein (HPF), and C11 BODIPY 581/591 were all purchased from (Shanghai Maokang Biotechnology, Shanghai, China).

[19] Fang, F., Wang, S., Song, Y. et al. Continuous Spatiotemporal Therapy of A Full-API Nanodrug via Multi-Step Tandem Endogenous Biosynthesis. Nat Commun 14, 1660 (2023). https://doi.org/10.1038/s41467-023-37315-0

FeRhoNox-1 (Fe2+ indicator) and hydroxyphenyl fluorescein (HPF) were purchased from Shanghai Maokang Biotechnology Co., Ltd.

[20] Hao Hu et al. Polydopamine-Pd nanozymes as potent ROS scavengers in combination with near-infrared irradiation for osteoarthritis treatment. April 2023 iScience 26(5):106605. DOI:10.1016/j.isci.2023.106605

The scavenging capacity of hydroxy radicals of PDA and PDA-Pd NPs at cellular levels was evaluated using the hydroxyl radical detection kit (hydroxyphenyl fluorescein, Maokangbio, China).

[21]Yin M, Lei D, Liu Y, Qin T, Gao H, Lv W, Liu Q, Qin L, Jin W, Chen Y, Liang H, Wang B, Gao M, Zhang J, Lu J. NIR triggered polydopamine coated cerium dioxide nanozyme for ameliorating acute lung injury via enhanced ROS scavenging. J Nanobiotechnology. 2024 Jun 8;22(1):321. doi: 10.1186/s12951-024-02570-w. PMID: 38849841; PMCID: PMC11162040.

Next, the medium was replaced with fresh staining mixtures containing 2’, 7’-dichlorofluorescin diacetate (DCFA, maokangbio, China) for total ROS testing, dihydroethidium (DHE, maokangbio, China) for ·O₂⁻ testing, and hydroxyphenyl fluorescein (HPF, maokangbio, China) for ·OH testing respectively by following the protocols before being imaged by fluorescent microscopy.

[22]Wu, Z., Zhang, P., Yue, J. et al. Tea polyphenol nanoparticles enable targeted siRNA delivery and multi-bioactive therapy for abdominal aortic aneurysms. J Nanobiotechnol 22, 471 (2024). https://doi.org/10.1186/s12951-024-02756-2

Hydroxyphenyl fluorescein (HPF) was obtained from Maokang Biotechnology Co., Ltd. (Shanghai, China). 

[23] Na An et al. Laser-induced breakdown spectroscopy-guided synergistic phototherapy: A theranostic platform mediated by a single laser wavelength. Colloids and Surfaces B: Biointerfaces. Volume 218, October 2022, 112768

Singlet oxygen sensor green (SOSG) and 3′-p-(hydroxyphenyl) fluorescein (HPF) were purchased from MaokangBio, Shanghai, China

[24] Huang, L., Yao, Y., Ruan, Z. et al. Baicalin nanodelivery system based on functionalized metal-organic framework for targeted therapy of osteoarthritis by modulating macrophage polarization. J Nanobiotechnol 22, 221 (2024). https://doi.org/10.1186/s12951-024-02494-5

The antioxidant capacity was assessed using the Reactive Oxygen Species Assay Kit (Beyotime, China). RAW264.7 cells were initially exposed to LPS (10 ng/ml) at 37℃ for 24 h, then treated with Bai and different NPs for another 24 h. Cells were then co-cultured with 10 µM HPF (maokangbio, China) for ·OH level testing, 5 µM DHE (Beyotime Biotechnology, China) for ·O₂⁻ level testing, and 20 µM DCF (Solarbio, China) for total ROS level testing.