FerroOrange (Fe2+indicator) 亚铁离子荧光探针

产品标签

FeRhoNox-1；FerroOrange；Labile ferrous ion不稳定铁(II)离子；Fe2+荧光探针；Ferroptosis铁死亡；C11 BODIPY 581/591脂质过氧化探针；

产品信息

产品描述

FerroOrange是一种特异性检测不稳定的铁(II)离子（Fe2+）的荧光探针，定位在内质网（ER）。一旦与Fe2+反应后，不可逆的生成一种橙色荧光产物（Absmax=542nm，FLmax=572nm，图1. FerroOrange的光谱特征）。铁（III）离子（Fe3+）或其它除铁离子以外的二价金属离子都不会使其荧光增强（见图2. FerroOrange的反应特异性）。FerroOrange也不会与铁蛋白和其它物质中的螯合铁反应。FerroOrange具强细胞膜渗透性和低细胞毒性（见附图3. FerroOrange的细胞毒性测定），非常适用于活细胞成像。

图1. FerroOrange的光谱特征。FerroOrange在0.05M HEPES buffer, pH 7.4内的吸收光谱（左）和荧光光谱。最大吸收波长在542nm，最大荧光光谱在572nm。一旦与Fe2+反应，荧光强度显著增加。

图2. FerroOrange的反应特异性。FerroOrange与各种金属离子（左），活性氧或还原剂（右）的反应性。仅当与Fe2+反应，FerroOrange呈强荧光。

保存与运输方法

保存：-20℃避光干燥保存，至少1年有效。

运输：冰袋运输。

注意事项

1. FerroOrange主要定位在内质网（见附图4），但也可能检测细胞质池内的Fe2+，目前针对这一点未做明确评估。

2. 荧光染料均存在淬灭问题，请尽量注意避光，以减缓荧光淬灭。

3. 为了您的安全和健康，请穿实验服并戴一次性手套操作。

使用说明

1. 需要自行准备的材料

1.1 细胞培养级DMSO（比如：MS4601A-100ML）

1.2 合适的清洗和观察缓冲液（比如：PBS, pH 7.4；HBSS；无血清培养基等）。不要含酚红。

2. 探针准备

2.1 从冰箱取出FerroOrange，置于室温至少30min，将其置于微量离心机内低速离心。将瓶内的粉末离心到管底后，再开盖。

2.2 往一管FerroOrange（24μg）内加入35μl高质量DMSO，用枪反复吹吸5次或以上，使其完全溶解即得到1mMFerroOrange储存液。若单次不能用完储存液，请根据单次用量分装，置于-20℃避光保存，一个月内使用。

2.3 于正式实验前，用中性缓冲液或细胞培养基来稀释1mM FerroOrange储存液到所需工作浓度（比如：1μM），工作液需现配现用，尽快用完。【注意：酸性溶液会氧化FerroOrange，严重影响探针的效率】。

3. 染色步骤

3.1于荧光培养皿中接种细胞，于37℃，5% CO2培养箱中孵育过夜。

3.2 染色步骤

4. 荧光检测

对于荧光显微镜：通用的G激发滤片比如Cy3检测用的滤片。

对于激光显微镜和流式细胞仪：532nm，514nm或561nm激光器用于激发。实在没有，亦可用488nm激光器。发射波长为572nm。

相关产品

附录 FerroOrange的细胞毒性

图3.各种浓度FerroOrange对HepG2细胞的代谢活性。不同浓度的FerroOrange（以1% DMSO作为共溶剂）加入细胞内，培养24h后，用MTT法测定代谢活性。（n=3）

附录FerroOrange的胞内定位

图4.FerroOrange的胞内定位。将FerroOrange（红色）, ER Tracker（绿色）和Hoechist 3342（蓝色）加载进入HT-1080细胞并成像观察。FerroOrange主要定位在内质网。Bar：10 μm。

 — —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。

引用文献：

[1]Yao, Y., Chen, Z., Zhang, H. et al. Selenium–GPX4 axis protects follicular helper T cells from ferroptosis. Nat Immunol 22, 1127–1139 (2021). https://doi.org/10.1038/s41590-021-00996-0

Cells were stained with FerroOrange (1 µM, MKBio, MX4559)

[2] Tang Z, Dong H, Li T, Wang N, Wei X, Wu H, Liu Y, Wang W, Guo Z, Xiao X. The Synergistic Reducing Drug Resistance Effect of Cisplatin and Ursolic Acid on Osteosarcoma through a Multistep Mechanism Involving Ferritinophagy. Oxid Med Cell Longev. 2021 Dec 21;2021:5192271. doi: 10.1155/2021/5192271. PMID: 34970416; PMCID: PMC8714329.

FerroOrange (Mkbio, Shanghai, China) was used to measure intracellular and mitochondrial Fe²⁺ levels according to the manufacturer's protocol. HOS cells were treated with Cis, UA, UA/Cis, UA/Cis/DFO, or UA/Cis/3-MA and stained with 1 μM FerroOrange for 30 min at 37°C. Images were acquired using a fluorescence microscope.

[3] Yi, Z.-H.; Li, S.-Q.; Ke, J.-Y.; Wang, Y.; Zhao, M.-Z.; Li, J.; Li, M.-Q.; Zhu, Z.-L. Baicalein Relieves Ferroptosis-Mediated Phagocytosis Inhibition of Macrophages in Ovarian Endometriosis. Curr. Issues Mol. Biol. 2022, 44, 6189-6204. https://doi.org/10.3390/cimb44120422

FerroOrange (Fe2+ indicator) (MKbio, Shanghai, China) was dissolved in dimethyl sulfoxide (DMSO) to form a 1 mM solution, which was further diluted (1:200) to 5 μM working concentration. Macrophages were seeded in 12-well plates with different treatments. The cells were then washed twice in PBS and incubated in 5 μM FerroOrange solution for 30 min at 37 °C avoiding light. Finally, the cells were rinsed in PBS twice and immediately observed.

[4] Ding X, Ma Y, Liu Q, Pang Y, Cao Y, Zhang T. How UV radiation and pH alternation impact graphene oxide mediated environmental toxicant adsorption and resulting safety characteristics - A toxicology study beyond a classic carrier effect. Chemosphere. 2022 Aug;300:134627. doi: 10.1016/j.chemosphere.2022.134627. Epub 2022 Apr 16. PMID: 35439484.

 The Fe 2+ level of L02 cells after treatment was detected by FerroOrange ferrous ion fluorescence probe (Maokang Bio-Technology Co., LTD, Shanghai, China). 

[5] Zhang L, Li D, Chang C, Sun Y. Myostatin/HIF2α-Mediated Ferroptosis is Involved in Skeletal Muscle Dysfunction in Chronic Obstructive Pulmonary Disease. International Journal of Chronic Obstructive Pulmonary Disease. 2022 ;17:2383-2399. DOI: 10.2147/copd.s377226. PMID: 36185172; PMCID: PMC9519128.

Cellular Fe²⁺ was detected by FerroOrange (MX4559, Maokangbio, Shanghai, China). FerrOrange is a fluorescent probe that specifically detects Fe²⁺ which is mainly located in the endoplasmic reticulum. Once FerroOrange reacts with Fe²⁺, it can generate an orange fluorescent product (Abs_max=542 nm, FL_max=572 nm) irreversibly. The C2C12 myotubes were washed 3 times in a serum-free medium and then 1 μM of FerrOrange was added to the medium for 30 min at 37°C. The level of Fe²⁺ was detected using a CytoFlex LX flow cytometer.

[6] Zeng F, Nijiati S, Liu Y, Yang Q, Liu X, Zhang Q, Chen S, Su A, Xiong H, Shi C, Cai C, Lin Z, Chen X, Zhou Z. Ferroptosis MRI for early detection of anticancer drug-induced acute cardiac/kidney injuries. Sci Adv. 2023 Mar 10;9(10):eadd8539. doi: 10.1126/sciadv.add8539. Epub 2023 Mar 8. PMID: 36888714; PMCID: PMC9995079.

FerroOrange was purchased from Maokang Biotechnology.

[7] Wu Y, Jia Q, Tang Q, Deng H, He Y, Tang F. Berberine-mediated Ferroptosis through System Xc^-/GSH/GPX4 Axis Inhibits Metastasis of Nasopharyngeal Carcinoma. J Cancer. 2024 Jan 1;15(3):685-698. doi: 10.7150/jca.90574. PMID: 38213727; PMCID: PMC10777030.

Fe²⁺ ions were detected as previously reported ³³. Briefly, the contents of Fe²⁺ in S18 and 5-8F cells were determined according to the manufacturer's instructions (MX4559-24UG; Shanghai Maokang Biotechnology Co.). FerroOrange (1 μM, an intracellular Fe²⁺ ions probe) dispersed in serum-free medium was added to the cells, and cells were incubated for 30 min in a 37 °C incubator, and cells were then collected. Finally, cell fluorescence was observed using a fluorescence microscope.

[8] Hao Wang et al. Metronomic capecitabine with rapamycin exerts an immunosuppressive effect by inducing ferroptosis of CD4+ T cells after liver transplantation in rat. International Immunopharmacology

Volume 124, Part A, November 2023, 110810

Rat CD4⁺ T cells were resuspended in phosphate-buffered saline (PBS) containing DCFH-DA  and incubated at 37 °C for 30 min, and intracellular ROS were detected by flow cytometry. Lipid peroxidation was measured by incubating cells with 2-μM BODIPY 581/591 C11 (Maokang Biotechnology, Shanghai, China) for 30 min at 37 °C and monitoring the signals from oxidized C11 (FITC channel). A JC-1 assay kit  was used to measure the mitochondrial membrane potential (MMP). Cells were incubated with JC-1 working solution at 37 °C for 30 min for fluorescence detection. Ferrous iron levels in rat peripheral blood and cultured T cells were detected by measuring fluorescence after incubating the cells at 37 °C with FerroOrange (Maokang Biotechnology, Shanghai, China) for 20 min.

[9] Sai Zhang et al. Ginkgolic acid inhibits the expression of SAE1 and induces ferroptosis to exert an anti-hepatic fibrosis effect. Phytomedicine. Volume 126, April 2024, 155148

FerroOrange (Maokang Biotechnology, China), an ROS assay kit (Zhang et al., 2022), C11-BODIPY 581/591 (Maokang Biotechnology, China), and JC-1 were used to detect the Fe²⁺levels, ROS levels, lipid ROS levels, and mitochondrial membrane potential in hepatic stellate cells. 

[10] Yi X, Long X, Liu C. Activating autophagy and ferroptosis of 3Chloropropane1,2diol induces injury of human umbilical vein endothelial cells via AMPK/mTOR/ULK1. Mol Med Rep. 2023 Mar;27(3):76. doi: 10.3892/mmr.2023.12963. Epub 2023 Feb 17. PMID: 36799162; PMCID: PMC9950850.

FerroOrange staining

A FerroOrange (Fe²⁺ indicator) probe (cat. no. MX4559; Shanghai Maokang Biotechnology Co., Ltd.) was used to assess intracellular Fe²⁺ levels according to the manufacturer's protocol. Briefly, cells were incubated with 1 µM FerroOrange for 30 min at 37°C. The cells were assessed using a fluorescence microscope (magnification, ×20; Carl Zeiss AG).

[11] Yang R, Gao W, Wang Z, Jian H, Peng L, Yu X, Xue P, Peng W, Li K, Zeng P. Polyphyllin I induced ferroptosis to suppress the progression of hepatocellular carcinoma through activation of the mitochondrial dysfunction via Nrf2/HO-1/GPX4 axis. Phytomedicine. 2024 Jan;122:155135. doi: 10.1016/j.phymed.2023.155135. Epub 2023 Oct 12. PMID: 37856990.

[12] Tang, Yj., Zhang, Z., Yan, T. et al. Irisin attenuates type 1 diabetic cardiomyopathy by anti-ferroptosis via SIRT1-mediated deacetylation of p53. Cardiovasc Diabetol 23, 116 (2024). https://doi.org/10.1186/s12933-024-02183-5

FerroOrange was purchased from the Maokang Institute of Biotechnology (Shanghai, China).