Hydroxyphenyl fluorescein (HPF) 

羥苯基熒光素  （金標產品，享發文獎勵）

產品信息

產品描述

氨基苯基熒光素（APF）和羥苯基熒光素（HPF）是由T. Nagano開發的兩種活性氧（Reactive oxygen species, ROS）指示劑，比H2DCFDA（DCFH-DA, DCFH）具更優秀的特異性和穩定性。APF和HPF顯示有限的非選擇性反應，和相對較高的光誘導氧化抗性。APF和HPF具細胞膜滲透性，本身無熒光，直到與羥基自由基或過氧亞硝基陰離子反應，還能與次氯酸陰離子反應，產生強綠色熒光（Ex/Em，~490/515nm），可用熒光顯微鏡、高通量成像儀、熒光酶標板或流式細胞儀檢測。

本品以粉末形式提供，可直接溶于DMF配制成5mM儲存液，常用工作濃度范圍1-10μM，需根據自身的實驗體系或參考文獻進行優化。

產品特性

1）CAS NO.：359010-69-8

2）同義名：3'-p-(hydroxyphenyl) fluorescein (HPF)羥苯基熒光素

3）分子式：C26H16O6

4）分子量：424.4

5）純度：≥95%

6）Ex/Em：492/515nm

7）溶解性：溶于DMSO（10mM）、DMF（5mM）

8）化學結構：

保存與運輸方法

保存：-20oC避光干燥保存，至少一年有效。

運輸：冰袋運輸。 

使用方法

一、儲存液制備

將低溫保存的1mg粉末置于室溫回溫至少20min，之后加入471μl 無水DMF配置成5mM儲存液。充分溶解和混勻后，按照單次用量分裝，-20oC避光干燥保存，避免反復凍融。

【注】：HPF能溶于DMSO（10mM），但有數據顯示DMSO是羥基自由基的淬滅劑，會干擾檢測和降低靈敏性。因此，建議用DMF作為溶劑。

【注】：與DMSO相比，DMF的細胞毒性相對較高，建議用戶在使用過程中，在達到理想熒光信號的前提下使用最低濃度的探針。如果待檢細胞在培養的過程中就發現比較敏感（或不好培養），建議在使用HPF之前，先做一組溶劑對照組來觀察細胞對DMF的敏感程度。

二、溶液體系檢測

正式實驗前，取一管HPF儲存液置于室溫回溫，使其充分融解。之后用合適的緩沖液（比如HHBS緩沖液，PBS緩沖液）稀釋到所需的工作濃度。建議起始濃度為1-10μM，需根據自身的實驗體系或參考文獻進行優化。

三、細胞體系檢測

以下實驗步驟僅做參考，最佳的工作濃度、孵育時間和溫度需根據實際情況進行調整。在加入HPF工作液之前按需處理細胞。

1）   根據具體用量，將DMF儲存液用HHBS緩沖液或其他生理緩沖液稀釋成1-10μM工作液。

2）   根據實驗設計處理細胞（比如，RASM細胞用50-100nM 血管緊張素II處理3-5h）。

3）   取步驟1）準備的1-10μM工作液加入細胞，37oC孵育20-60min。

4）   吸掉染色工作液，用HHBS或其他生理緩沖液替換。

5）   用合適的熒光儀器（Ex/Em=492/515nm）來檢測。

【注1】：BSA和酚紅會影響熒光，需謹慎使用。

【注2】：HPF和APF能用于溶液體系或胞內ROS檢測。

注意事項

1）   APF和HPF僅用于科研研究，不可用于體外診斷研究。

2）   APF和HPF產生的熒光相對于其他活性氧探針比如DCFH弱很多，這是探針本身的特性決定的（見附表1 活性氧探針（HPF, APF, DCFH）對各種活性氧物質的反應特異性）。

3）   為了您的安全和健康，請穿實驗服并戴一次性手套操作。

相關產品

附表1 活性氧探針（HPF, APF, DCFH）對各種活性氧物質的反應特異性

 — —Written/Edited by V. Shallan【版權歸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學和生物技術領域研究的試劑、儀器和實驗室消耗品與實驗服務工作，主要從事細胞生物學、植物學、分子生物學、免疫學、生物化學、蛋白組學。生物制藥與診斷試劑研發生產等領域。 本公司秉承“以人為本，以誠為信、合同守信”的經營理念。堅持"品質保障"的原則為廣大客戶提供優質產品。

文獻引用：

[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.