BODIPY 665/676 脂质过氧化荧光探针

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描述

BODIPY 665/676 脂质过氧化荧光探针

 

产品关键词

BODIPY 665/676；C11 BODIPY 581/591；Lipid Peroxidation Sensor；脂质过氧化感受器；铁死亡（Ferroptosis）；FeRhoNox-1 (Fe2+ indicator) 亚铁离子荧光探针；

产品信息

产品名称

 产品编号 规格       价格（元） 
BODIPY 665/676脂质过氧化荧光探针 MX5212-5MG 5mg

3593

产品描述

BODIPY 665/676是一种亲脂性荧光探针，高亲和力结合游离脂肪酸和甘油三酯，具有长波长的最大吸收波长（λex=665nm）和最大荧光发射波长（Em=676nm）[1]。与常用的脂质过氧化探针C11 BODIPY 581/591（MX5211-1MG）相似，BODIPY 665/676容易被过氧化自由基氧化，一旦氧化，最大发射波长迁移到605nm（λex =580 nm）[2-4]。BODIPY 665/676最重要的优势在于发射波长是676nm，与绝大部分的荧光素之间无交叉，从而能避免比如自荧光的干扰。BODIPY 665/676具热稳定和高度疏水性特征，从而不会扩散出脂滴。激发器642nm与最大吸收波长665nm接近，由此，在很低的探针浓度下就能得到强荧光信号[5]。

产品特征

1）   同义名：(E,E)-3,5-bis-(4-phenyl-1,3-butadienyl)-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene

2）   分子量：448.32

3）   外观：蓝色固体

4）激发波长：665nm

5）发射波长：676nm

保存与运输方法

保存：-20ºC避光干燥保存，收到货至少12个月有效。

运输：冰袋运输。

注意事项

1）   整个染色过程中需注意避光。

2）   为了您的安全和健康，请穿实验服并戴一次性手套操作。

使用方法（活细胞成像分析）

1.染色工作液的准备

1.1储存液的制备：将低温保存的BODIPY 665/676干粉从冰箱取出后置于室温至少20min，低速离心后，加入高质量DMSO配制成适宜浓度的储存液，比如：10mM储存液（即：往5mg BODIPY 665/676加入1.1153ml DMSO，充分溶解后混匀即可），根据单次用量分装，≤-20℃保存，尽量避免反复冻融。

1.2工作液的制备：BODIPY 665/676用于活细胞染色的建议工作浓度一般是1-10 μM，请根据细胞类型和实验目的，参阅文献，再根据实际应用摸索最佳工作浓度。用培养基或生理缓冲液稀释10mM储存液到所需的工作浓度，现配现用！

2.染色步骤

3.图像处理和分析

应用示例（来自文献，仅做参考）

1）   为了测定脂质过氧化（Lipid Peroxidation），MDA-MB 231细胞加入BODIPY 665/676（5 μM）孵育30 min。用PBS清洗细胞两次以去除多余的染料。用VariosKan酶标仪测定荧光值，结果以荧光强度（AU）来展示。【文献来源】：Consoli V, Sorrenti V, Pittalà V, Greish K, D’Amico AG, Romeo G, Intagliata S, Salerno L, Vanella L. Heme Oxygenase Modulation Drives Ferroptosis in TNBC Cells. Int J Mol Sci. 2022 May 20;23(10):5709. doi: 10.3390/ijms23105709. PMID: 35628518; PMCID: PMC9143660.

Fig 1. (A) Fluorescence images of JC-1 staining after 8 h of treatment. (B,C) Evaluation of treatment effects on mitochondrial membrane potential and lipid ROS accumulation (* p < 0.05, ** p < 0.005, vs. CTRL; ## p < 0.005, ### p < 0.0005 vs. erastin). The results are expressed as means ± SEM.

2）   为了评估中性脂滴含量和脂质过氧化，细胞用BODIPY 665/676（1μg/ml）于37℃孵育30 min。孵育结束后，细胞用FACSCalibur流式细胞仪分析。【文献来源：Canonico B, Cesarini E, Salucci S, Luchetti F, Falcieri E, Di Sario G, Palma F, Papa S. Defective Autophagy, Mitochondrial Clearance and Lipophagy in Niemann-Pick Type B Lymphocytes. PLoS One. 2016 Oct 31;11(10):e0165780. doi: 10.1371/journal.pone.0165780. PMID: 27798705; PMCID: PMC5087958.】

Fig 2. Fig 6. Evaluation of intracellular lipid content and lipophagy by BODIPY® 665/676 (BP).

(A) Single confocal optical sections (~0.8 μm thickness) showing overlay of LTG (green) and BP (red) and the relative merge with bright field (BF), from control, nutrient deprivation and rapamycin- treated tWT and tNP cells. Insets show lipid droplets bound to the outer cell surface, probably newly extruded from the cell, or just below the outer cell membrane, most likely about to be exocytosed by the cell. Bars: 10 μm; 5 μm for insets. (B) Pearson’s colocalization coefficient (PCC) for LTG and BP in control and pathologic cells for control, nutrient deprivation and rapamycin administration. Pearson’s coefficients were derived from three completely independent experiments with >10 fields per experiment contributing to the cumulative result. Each value is expressed as PCC ± SD; **P < 0.01 vs respective control. The difference between cell lines was determined to be significant by two-way ANOVA (***P < 0.001). Two-way ANOVA with Bonferroni post test revealed a P value < 0.01 (++P < 0.01) between tWT and tNP basal condition and rapamycin-treated cells. (C) Statistical histogram of MFI variation of intracellular BP in tWT and tNP cells for each experimental condition. Mean values were converted to arbitrary units (A.U.) setting control of wild-type cells as 100. Each value is expressed as a relative mean ± SD (Results from n ≥ 3 independent experiments); **P < 0.01 vs respective control.

3）为了测定脂质氧化（lipid Peroxides），细胞用总内皮细胞处理，之后用BODIPY 665/676（2μg/ml）于37℃孵育30 min。之后上流式，用FlowJo 10.0.0软件进行数据分析。【文献来源：Rodrigues, J., Wang, YF., Singh, A. et al. Estrogen enforces the integrity of blood vessels in the bone during pregnancy and menopause. Nat Cardiovasc Res 1, 918–932 (2022). https://doi.org/10.1038/s44161-022-00139-0】

参考文献

[1] Canonico B, Cesarini E, Salucci S, Luchetti F, Falcieri E, Di Sario G, et al. (2016) Defective Autophagy, Mitochondrial Clearance and Lipophagy in Niemann-Pick Type B Lymphocytes. PLoS ONE 11(10): e0165780.

[2] Meyer W, Schmidt J, Busche R, Jacob R, Naim HY. Demonstration of lipids in plastic resin-embedded sections of skin material. J Microsc. 2009; 233: 5–9. pmid:19196406

[3] Raudsepp P, Brüggemann DA, Andersen ML. Detection of radicals in single droplets of oil-in-water emulsions with the lipophilic fluorescent probe BODIPY(665/676) and confocal laser scanning microscopy. Free Radic Biol Med. 2014 May;70:233-40. doi: 10.1016/j.freeradbiomed.2014.02.026. Epub 2014 Mar 12. PMID: 24631488.

[4] Raudsepp P, Brüggemann DA, Andersen ML. Evidence for transfer of radicals between oil-in-water emulsion droplets as detected by the probe (E,E)-3,5-bis(4-phenyl-1,3-butadienyl)-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene, BODIPY(665/676.). J Agric Food Chem. 2014 Dec 24;62(51):12428-35. doi: 10.1021/jf504404a. Epub 2014 Dec 12. PMID: 25440428.

[5] Li, Peilong, “Application of Flow Cytometry as Novel Technology in Studying Lipid Oxidation in Oil-in-Water Emulsions” (2019). Masters Theses. 843.

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