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Phen Green SK diacetate

(Synonyms: 苯绿SK二乙酸酯,PGSK diacetate) 目录号 : GC40243

Phen Green SK diacetate (PGSK)是一种重金属荧光指示剂,对Fe2+、Cd2+、Co2+、Ni2+和Zn2+等一系列金属离子具有反应性。

Phen Green SK diacetate Chemical Structure

Cas No.:234075-45-7

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Sample solution is provided at 25 µL, 10mM.

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实验参考方法

Protocol for characterization of copper transport on the plasma membrane of CSB cells [1]:

10 mM Phen Green SK storage solution was prepared with DMSO, and the storage solution was stored away from light at -20℃ or -80℃ after subpackaging, and used up within 2 weeks.

Preparation of 80µM Phen Green SK working solution was prepared by preheating serum-free cell medium or PBS. Please adjust the concentration of the working liquid according to the actual situation.

1.Flow cytometry experiments

Intact protoplasts isolated from both 0.1 µM (control) and 100µM CuSO4-grown cells were labeled with the copper-sensitive probe Phen Green SK diacetate (PGSK) to demonstrate copper sequestration.

 Vacuoles were isolated from PGSK-loaded protoplasts to see if the probe could enter this organelle.

Samples were analyzed in flow cytometer equipped with an argon-ion laser emitting a 488 nm beam at 15 mW.

Green fluorescence was collected through a 488 nm blocking filter, a 550 nm long-pass dichroic filter and a 525 nm band-pass filter. For each sample, 5,000 protoplasts or vacuoles were analyzed at low flow rate.

2.Copper transport by CSB protoplasts with Phen Green SK diacetate

Copper transport across the plasma membrane was determined with PGSK using spectrofluorometer with excitation and emission wavelengths set at 506 and 532 nm, respectively.

For transport experiments, protoplasts (106) were incubated with 80µM PGSK for 30 min to equilibrate the dye.

The loaded protoplasts (100 µl) were added to the assay cuvette with 9 vols. of the mannitol-containing buffer, pH 5.6. After stabilization of the fluorescence signal, different amounts of CuCl2 were added (0.1-3.5 mM, final concentrations).

To discriminate the unspecific fluorescence quenching and the quenching derived from copper uptake by protoplasts, the chelators EDTA and BCS were added to the assay medium. The effect of Cu2+ on the hydrolyzed external probe was tested after treatment of PGSK-loaded protoplasts with 0.5% Triton X-100.

To study which form of copper ion was taken up by protoplasts, ascorbic acid was added to the assay medium, reducing Cu2+ to Cu+.

To assess the effect of various cations on Cu2+ uptake, protoplasts were incubated for 5 min with the chloride forms of Ca2+ (10 mM), K+ (10 mM), Mn2+ (5 mM), Na+ (10 mM) and Zn2+ (10 mM) before the addition of CuCl2.

To test how Cu2+ uptake is regulated by copper availability in the medium, cells grown for 6 d were incubated for 8 h with 100 µM CuSO4 or with 20 µM of the chelator BCS.

Protocol for measure intracellular ferrous iron levels [2]:

1.Cells were treated as indicated before 10 μM Phen Green SK diacetate was added and incubated for 10 min.

2.The cells were washed twice with PBS to remove the excess PGSK.

3.The cells were trypsinized and resuspended in PBS plus 5% FBS.

4.A flow cytometer was used for detection of ferrous iron. Each group was triplicated.

 

This protocol only provides a guideline, and should be modified according to your specific needs.

References:

[1].  Martins V, Hanana M, et,al. Copper transport and compartmentation in grape cells. Plant Cell Physiol. 2012 Nov;53(11):1866-80. doi: 10.1093/pcp/pcs125. Epub 2012 Sep 5. PMID: 22952251.

[2].  Wang Z, Yao M, et,al. Dexmedetomidine attenuates myocardial ischemia/reperfusion-induced ferroptosis via AMPK/GSK-3β/Nrf2 axis. Biomed Pharmacother. 2022 Oct;154:113572. doi: 10.1016/j.biopha.2022.113572. Epub 2022 Aug 18. PMID: 35988428.

产品描述

Phen Green SK diacetate (PGSK) represents a fluorescent indicator for heavy metals, demonstrating reactivity towards a range of metal ions encompassing Fe2+, Cd2+, Co2+, Ni2+, and Zn2+. PGSK is more sensitive to quenching by the ferrous ion (Fe2+) than by the ferric ion (Fe3+)[1].The excitation/emission peaks of PGSK diacetate are recorded at 507/532 nm, correspondingly, with its fluorescence being suppressed upon interaction with metal ions. Phen Green SK is a membrane-impermeable fluorophore, sensitive only to free (chelatable) iron, which can be loaded into intact cells[2-4]. This compound has found applications in quantifying iron levels in isolated rat hepatocytes, elucidating the metal ion preferences of divalent metal-ion transporter 1 (DMT1), and tracking copper transportation across P. sativum chloroplast membranes and cells[5-7].

References:
[1]. Shingles R, North M, et,al. Direct measurement of ferrous ion transport across membranes using a sensitive fluorometric assay. Anal Biochem. 2001 Sep 1;296(1):106-13. doi: 10.1006/abio.2001.5209. PMID: 11520038.
[2].Illing AC, Shawki A, et,al.Substrate profile and metal-ion selectivity of human divalent metal-ion transporter-1. J Biol Chem. 2012 Aug 31;287(36):30485-96. doi: 10.1074/jbc.M112.364208. Epub 2012 Jun 26. PMID: 22736759; PMCID: PMC3436370.
[3]. Petrat F, de Groot H, et,al. Determination of the chelatable iron pool of single intact cells by laser scanning microscopy. Arch Biochem Biophys. 2000 Apr 1;376(1):74-81. doi: 10.1006/abbi.2000.1711. PMID: 10729192.
[4]. Rauen U, Petrat F, et,al.Hypothermia injury/cold-induced apoptosis--evidence of an increase in chelatable iron causing oxidative injury in spite of low O2-/H2O2 formation. FASEB J. 2000 Oct;14(13):1953-64. doi: 10.1096/fj.00-0071com. PMID: 11023979.
[5]. Petrat F, Rauen U, et,al.Determination of the chelatable iron pool of isolated rat hepatocytes by digital fluorescence microscopy using the fluorescent probe, phen green SK. Hepatology. 1999 Apr;29(4):1171-9. doi: 10.1002/hep.510290435. PMID: 10094962.
[6]. Shingles R, Wimmers LE, et,al.Copper transport across pea thylakoid membranes. Plant Physiol. 2004 May;135(1):145-51. doi: 10.1104/pp.103.037895. Epub 2004 Apr 30. PMID: 15122011; PMCID: PMC429342.
[7]. Martins V, Hanana M, et,al. Copper transport and compartmentation in grape cells. Plant Cell Physiol. 2012 Nov;53(11):1866-80. doi: 10.1093/pcp/pcs125. Epub 2012 Sep 5. PMID: 22952251.

Phen Green SK diacetate (PGSK)是一种重金属荧光指示剂,对Fe2+、Cd2+、Co2+、Ni2+和Zn2+等一系列金属离子具有反应性。PGSK对亚铁离子(Fe2+)比三铁离子(Fe3+)更敏感[1]。Phen green SK diacetate激发/发射最大值分别为507/532 nm。它作为一种膜不渗透荧光团,仅对游离(可螯合)铁敏感,可被导入到完整细胞中[2-4]。该化合物已被用于定量分离大鼠肝细胞中的铁水平,阐明二价金属离子转运体1 (DMT1)的金属离子偏好,以及追踪铜在豌豆叶绿体膜和细胞中的运输[5-7]

Chemical Properties

Cas No. 234075-45-7 SDF
别名 苯绿SK二乙酸酯,PGSK diacetate
化学名 3',6'-bis(acetyloxy)-2',7'-dichloro-3-oxo-N-1,10-phenanthrolin-5-yl-spiro[isobenzofuran-1(3H),9'-[9H]xanthene]-5-carboxamide
Canonical SMILES CC(OC1=CC2=C(C=C1Cl)C3(C(C=CC(C(NC4=CC(C=CC=N5)=C5C6=C4C=CC=N6)=O)=C7)=C7C(O3)=O)C8=CC(Cl)=C(OC(C)=O)C=C8O2)=O
分子式 C37H21Cl2N3O8 分子量 706.5
溶解度 DMSO : 50 mg/mL (70.77 mM; Need ultrasonic) 储存条件 Store at -20°C
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1 mM 1.4154 mL 7.0771 mL 14.1543 mL
5 mM 0.2831 mL 1.4154 mL 2.8309 mL
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Research Update

Baicalein and luteolin inhibit ischemia/reperfusion-induced ferroptosis in rat cardiomyocytes

Int J Cardiol 2023 Mar 15;375:74-86.PMID:36513286DOI:10.1016/j.ijcard.2022.12.018

Background: Ischemia/reperfusion (I/R) is associated with severe cellular damage and death. Ferroptosis, a new form of regulated cell death caused by the accumulation of iron-mediated lipid peroxidation, has been found in several diseases including I/R injury, which was reported to be suppressed by flavonoids. Baicalein (BAI) and luteolin (Lut) are flavonoids and were shown to reduce the myocardial I/R injury. BAI was found to suppress ferroptosis in cancer cells via reducing reactive oxygen species (ROS) generation. However, the anti-ferroptosis effect of Lut on ferroptosis has not been reported. This study aimed to investigate whether ferroptosis reduction contributes to the BAI- and Lut-protected cardiomyocytes. Methods: This research used erastin, RSL3, and Fe-SP to induce ferroptosis. Cell viability was examined using MTT assay. Annexin V-FITC, CM-H2DCFDA, and Phen Green SK diacetate (PGSK) fluorescent intensity were detected to analyze apoptotsis, ROS levels, and Fe2+ concentrations, respectively. qPCR and Western blot analysis were conducted to detect the levels of mRNA and protein, respectively. Results: Our data show that BAI and Lut protected cardiomyocytes against ferroptosis caused by ferroptosis inducers and I/R. Moreover, both BAI and Lut decreased ROS and malondialdehyde (MDA) generation and the protein levels of ferroptosis markers, and restored Glutathione peroxidase 4 (GPX4) protein levels in cardiomyocytes reduced by ferroptosis inducers. BAI and Lut reduced the I/R-induced myocardium infarction and decreased the levels of Acsl4 and Ptgs2 mRNA. Conclusions: BAI and Lut could protect the cardiomyocytes against the I/R-induced ferroptosis via suppressing accumulation of ROS and MDA.