FeRhoNox-1 (Fe2+indicator) 亞鐵離子熒光探針

產品標簽

FeRhoNox-1；FerroOrange；Labile ferrous ion 不穩定鐵(II)離子；Fe2+熒光探針；Ferroptosis 鐵死亡；C11 BODIPY 581/591 脂質過氧化探針；

產品信息

產品描述

FeRhoNox-1，也稱為RhoNox-1，是一種活躍的熒光探針，特異性檢測不穩定的鐵(II)離子（Fe2+）。一旦與Fe2+反應后，不可逆的生成一種橙色（紅色）熒光產物（Absmax=540nm，FLmax=575nm，圖1.FeRhoNox-1的光譜特征）。生理濃度下的鐵（III）離子（Fe3+）或其它除鐵離子以外的二價金屬離子都不會使其熒光增強（見圖2FeRhoNox-1的選擇性）.FeRhoNox-1的反應特異性）。FeRhoNox-1具細胞膜滲透性和高選擇性，適用于活細胞內Fe2+的檢測，傾向定位在高爾基體。

圖1. FeRhoNox-1的光譜特征。FeRhoNox-1與Fe2+反應后吸收和發射光譜（上）。FeRhoNox-1在37℃，與Fe2+反應1h后，熒光明顯增強。最大熒光峰約在575nm。

圖2. FeRhoNox-1的選擇性。FeRhoNox-1僅與Fe2+反應。

保存與運輸方法

保存：-20℃避光干燥保存，至少1年有效。

運輸：冰袋運輸。

注意事項

1. FeRhoNox-1傾向定位在高爾基體，但也可能檢測細胞質池內的Fe2+，目前針對這一點未做明確評估。

2. 熒光染料均存在淬滅問題，請盡量注意避光，以減緩熒光淬滅。

3. 為了您的安全和健康，請穿實驗服并戴一次性手套操作。

使用說明

1. 需要自行準備的材料

1.1細胞培養級或超純DMSO（比如：MS4601A-100ML）【強烈建議將高純的DMSO分裝成單次用量保存在極低溫冷凍室內，比如-80℃，避免吸潮。降解的DMSO可能會增加FeRhoNox-1的背景信號】

1.2合適的清洗和觀察緩沖液（比如：PBS, pH 7.4；HBSS；等）。不要含酚紅。

2. 探針準備

2.1從冰箱取出FeRhoNox-1，置于室溫回溫至少30min，將其置于微量離心機內低速離心。將瓶內的粉末離心到管底后，再開蓋。

2.2往一管FeRhoNox-1（50μg）內加入109μl高質量DMSO，用槍反復吹吸5次或以上，使其完全溶解即得到1mMFeRhoNox-1儲存液。建議單次用完儲存液，若實在用不完，請根據單次用量分裝，置于-80℃避光保存。用中性緩沖液來稀釋儲存液。

2.3于正式實驗前，用HBSS或其他中性緩沖液來稀釋1mMFeRhoNox-1儲存液到所需工作濃度（比如：5μM），工作液需現配現用，盡快用完?！咀⒁猓核嵝匀芤簳趸疐eRhoNox-1，嚴重影響探針的效率】。

3. 染色步驟

4. 熒光檢測

對于熒光激發：通用的綠色激發濾片比如Cy3或四甲基羅丹明（TMR）檢測用的濾片。

對于激光激發：532nm或543nm激光器比較適合。發射波長為570nm左右。

相關產品

附錄F eRhoNox-1的染色示例

I. HepG2細胞

圖3. FeRhoNox-1在HepG2活細胞內的成像檢測。①-Fe(II)：細胞未添加亞鐵離子（100μM硫酸亞鐵銨）；②+Fe(II)：細胞加載亞鐵離子；③+Fe(II)+Bpy：細胞加載亞鐵離子，之后用加入鐵離子螯合劑Bpy。圖②熒光增強，而圖③熒光降低。此結果與FeRhoNox-1特異性檢測Fe(II)的特征一致。

 — —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。

引用文獻：

[1]

Lu Zhou, Peng Yu, Ting-ting Wang, Yi-wei Du, Yang Chen, Zhen Li, Man-lin He, Lan Feng, Hui-rong Li, Xiao Han, Heng Ma, Hong-bao Liu, "Polydatin Attenuates Cisplatin-Induced Acute Kidney Injury by Inhibiting Ferroptosis", Oxidative Medicine and Cellular Longevity, vol. 2022, Article ID 9947191, 14 pages, 2022. https://doi.org/10.1155/2022/9947191 

Method: FeRhoNox-1 fluorescent probe (MX4558) was purchased from Maokang Biotech (Shanghai, China).FeRhoNox-1,which is a turn-on fluorescent probe specific for the detection of labile iron Fe2+, was used to detect intracellular LIP, and the cellular distribution of FeRhoNox-1 was consistent with Golgi [41]. HK-2 cells were grown to confluence in 35?mm laser confocal petri dishes in DMEM, and PD (40?μM) or Fer-1 (1?μM) was added in the absence or presence of cisplatin (20?μM). Cells were incubated with 5?μM FeRhoNox-1 for 1?h prior to assays. Cells were washed twice with PBS before staining nuclei with Hoechst 33342. The fluorescence was immediately observed with a confocal laser-scanning microscope (CLSM, ECLIPSE Ti, Nikon, Tokyo, Japan).

[2]

Jin R, Yang R, Cui C, Zhang H, Cai J, Geng B, Chen Z. Ferroptosis due to Cystathionine γ Lyase/Hydrogen Sulfide Downregulation Under High Hydrostatic Pressure Exacerbates VSMC Dysfunction. Front Cell Dev Biol. 2022 Feb 3;10:829316. doi:

10.3389/fcell.2022.829316. PMID: 35186934; PMCID: PMC8850391. 

Method: For FeRhoNox-1 staining, 5 μM of the FeRhoNox-1 staining working solution (MX4558 and MKBIO)was added and incubated in a 37°C, 5% CO2 incubator for 60 min after treatment for 24 h in a hydrostatic pressure chamber. After washing three times with PBS, cells were imaged with a confocal microscope.

[3] Zhang X, Ma Y, Ma J, Yang L, Song Q, Wang H, Lv G. Glutathione Peroxidase 4 as a Therapeutic Target for Anti-Colorectal Cancer Drug-Tolerant Persister Cells. Front Oncol. 2022 Jun 3;12:913669. doi: 10.3389/fonc.2022.913669. PMID: 35719967; PMCID: PMC9203854.

Method: FeRhoNox-1 (an Fe2+ indicator, Cat# MX4558) was purchased from MKBio (Shanghai, China). Ferrous Iron Staining

Cells were incubated for 1 h at 37°C with FeRhoNox-1 (an Fe2+ indicator) to detect ferrous iron. The cells were then harvested by trypsinization, and the level of ferrous iron was determined by imaging using a confocal microscope or by flow cytometry analysis.

[4] Fang J, Yuan Q, Du Z, Fei M, Zhang Q, Yang L, Wang M, Yang W, Yu J, Wu G, Hu J. Ferroptosis in brain microvascular endothelial cells mediates blood-brain barrier disruption after traumatic brain injury. Biochem Biophys Res Commun. 2022 Sep 3;619:34-41. doi: 10.1016/j.bbrc.2022.06.040. Epub 2022 Jun 14. PMID: 35728282.

Method: Intracellular Fe 2+ levels were evaluated by using FeRhoNox-1 probe (MKBio, Shanghai). 24 h after SI or RSL3 incubation, culture medium was washed by PBS and exchanged for HBSS

 Lai Y, Zeng F, Chen Z, et al. Shikonin Could Be Used to Treat Tubal Pregnancy via Enhancing Ferroptosis Sensitivity. Drug Design, Development and Therapy. 2022 ;16:2083-2099. DOI: 10.2147/dddt.s364441. PMID: 35800255; PMCID: PMC9255906.

Method: Labile Iron Pool (LIP) Assay

“Labile iron” (which is primarily in the ferrous (Fe2+) form) is a small, transitional pool of intracellular iron, and commonly termed “LIP”. LIP release was measured using a FeRhoNox-1? (Fe2+ indicator) fluorescent probe (MKBio, Beijing, China). HTR-8/SVneo cells were plated in six-well plates, loaded with FeRhoNox-1 (5 μM) for 30 min at 37°C and then washed thrice with Hanks’ balanced salt solution. Cells were observed under a fluorescence microscope (Olympus).

[5] Cui J, Zhou Q, Yu M, Liu Y, Teng X, Gu X. 4-tert-butylphenol triggers common carp hepatocytes ferroptosis via oxidative stress, iron overload, SLC7A11/GSH/GPX4 axis, and ATF4/HSPA5/GPX4 axis. Ecotoxicol Environ Saf. 2022 Sep 1;242:113944. doi: 10.1016/j.ecoenv.2022.113944. Epub 2022 Aug 1. PMID: 35926411.

[6] 

Method:  Intracellular Fe2+ determination

FeRhoNox-1 (Fe2+ Indicator) fluorescent probe (Maokang Biotechnology Co., Ltd., Shanghai, China) was used to detect intracellular Fe2+ content. The cells inoculated in cell culture dishes were treated separately (Please see 2.9 for details), and then were washed twice with PBS. The FeRhoNox-1 (5 μM) was added in the medium and the cells were incubated for 60 min. Finally, images were obtained under the fluorescence microscope, and Image J version 1.43 u software was used to quantify the fluorescence intensity.

{7]Hong H, Lin X, Xu Y, Tong T, Zhang J, He H, Yang L, Lu Y, Zhou Z. Cadmium induces ferroptosis mediated inflammation by activating Gpx4/Ager/p65 axis in pancreatic β-cells. Sci Total Environ. 2022 Nov 25;849:157819. doi: 10.1016/j.scitotenv.2022.157819. Epub 2022 Aug 2. PMID: 35931150.

Method: For cellular Fe 2+ detection, cells were stained with 5 μmol/L FeRhoNox-1 (Fe 2+ indicator, Maokangbio, China) for 60 min at incubator.

[8]

Hu Q, Zuo T, Deng L, Chen S, Yu W, Liu S, Liu J, Wang X, Fan X, Dong Z. β-Caryophyllene suppresses ferroptosis induced by cerebral ischemia reperfusion via activation of the NRF2/HO-1 signaling pathway in MCAO/R rats. Phytomedicine. 2022 Jul 20;102:154112. doi: 10.1016/j.phymed.2022.154112. Epub 2022 Apr 22. PMID: 35550220.

Method:The iron level in astrocytes was detected through FeRhoNox-1 (Fe 2+ indicator) (MX4588, Maokangbio, China)