FM 4-64 (N-(3-Triethylammoniumpropyl)-4-(6-(4-(Diethylamino) Phenyl) Hexatrienyl) Pyridinium Dibromide)

发表于由

FM 4-64；FM 1-43；SynaptoRed C2；Lipophilic probe；Yeast vacuolar membranes 酵母液泡膜；Synaptic vesicle 突触小泡； 

产品名称

 产品编号 规格
FM 4-64 (N-(3-Triethylammoniumpropyl)-4-(6-(4-(Diethylamino) Phenyl) Hexatrienyl) Pyridinium Dibromide)

MX4016-100UG

 100μg
MX4016-200UG 2×100μg

MX4016-1MG 1mg

 FM 4-64，英文全名：N-(3-Triethylammoniumpropyl)-4-(6-(4-(Diethylamino) Phenyl) Hexatrienyl) Pyridinium Dibromide，是一种亲脂的苯乙烯染料，用作一种活细胞探针示踪酵母整体膜内在化和运输到液泡。FM 4-64是一种灵敏的液泡动力学分析探针，检测一系列相关事件，包括有丝分裂中的分离结构形成、液泡分裂和融合事件，以及液泡蛋白质分拣蛋白突变体在不同阶段的液泡形态。

FM 4-64具水溶性、对细胞无毒性，在水溶液中基本无荧光，一旦插入脂膜表面发出强的红色荧光（Ex/Em= 558/734nm）。FM 4-64与呈绿色荧光的FM 1-43（MX4014）在合适的滤片能够区分开，允许双色实时观察膜的再循环。

产品特性

1）同义名：N-(3-Triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide

2）分子式：C30H45Br2N3

3） 分子量：607.51

4） 纯度：≥90%（HPLC）

5） 外观：紫色固体 

6） 溶解性：溶于水、DMSO

7） Ex/Em：558/734nm（与膜结合）

8） 溶解性：溶于水

9） 化学结构式：

保存与运输方法

保存：-20°C避光干燥保存，至少1年有效。 

运输：冰袋运输。

注意事项

  • 荧光染料都存在淬灭的问题，保存和操作过程中注意避光。
  • FM®是Molecular Probe公司的注册商标。
  • 为了您的安全和健康，请穿实验服并戴一次性手套操作。

应用示例（来自文献，仅作参考）

文献一、Luo Wj et al. Novel Genes Involved in Endosomal Traffic in Yeast Revealed by Suppression of a Targeting-defective Plasma Membrane ATPase Mutant. J Cell Biol. 1997 Aug 25;138(4):731-46. PMID: 9265642

使用方法（FM 4-64 Endocytosis）：For FM 4-64 internalization studies, cells were grown to mid-log phase in YPD. After resuspension in fresh YPD at 20 OD600/ml, 20 μM FM 4-64 was added for 5 min at 30°C. Cells were washed, and incubation continued at 30°C for 1 h. FM 4-64 fluorescence was observed with rhodamine fluorescence filter sets. 【使用FM 4-64的原理在于：FM 4-64是批量质膜内在化的一种标记探针，从细胞表面到液泡膜的内吞示踪的转运以一种时间、温度和能量依赖性的方式进行。】

Fig 1. Visualization of endocytosis in sop mutants by FM 4-64 staining. Exponentially growing wild-type cells and sop mutants were stained with FM 4-64 for 5 min at 30°C, washed, and incubated in fresh YPD for 1 h before visualization and photography. Vacuolar membrane staining is seen in wild-type cells (L3852). In vps36 (WLX12-7C), a class E vps mutant, FM 4-64 dye is accumulated in a prevacuolar compartment. A similar accumulation of dye as a spot near the vacuole is seen in vps38 (WLX14-10A) and vps13 (WLX15-4C). Bright punctate staining in the cytoplasm remains in vps8 (WLX16-1A) after 1 h of chase.

文献二、Masamitsu Shimazu et al. A Family of Basic Amino Acid Transporters of the Vacuolar Membrane from Saccharomyces cerevisiae.

使用方法（FM 4-64 stain for vacuolar membrane）：Subcellular localization of Vba1p-GFP fusion protein in living S. cerevisiae cells was assessed using fluorescence microscopy. To stain vacuolar membranes, FM 4–64 was added to growing cultures to a final concentration of 5 μM. The cells were further cultured for 20 min and harvested. After washing, the cells were resuspended in fresh YPD media for 30 min to allow the dye to stain the vacuole via endocytosis.

FIG 2. Fluorescence microscopy of the transformant Δvba1/pVBA1GFP. A, GFP fluorescence; B, FM 4–64 fluorescence; C, Nomarski; D, merged image.

文献三、Sun Y et al. Scribble interacts with beta-catenin to localize synaptic vesicles to synapses. Mol Biol Cell. 2009 Jul;20(14):3390-400.

使用方法（FM 4-64 stain for presynaptic terminals）：Briefly, 15 μM FM 4-64 was loaded for 30 s into presynatpic terminals using a hyperkalemic solution of 90 mM KCl in modified HBSS, where equimolar NaCl was omitted for final osmolality of 310 mOsm. Neurons were rinsed three times and maintained in HBSS without Ca2+ for imaging. ADVESAP-7 (1 mM) was added to quench the nonspecific signal. Three images were captured every 30 s to confirm that the positive FM 4-64 sites were stationary presynaptic terminals. Unloading was done using the hyperkalemic solution described above, and neurons were rinsed three times with NeuroBasal media for continued imaging.

Fig 3. Deficits in SV recycling after scribble knockdown. (A–F) Confocal images of 10 DIV neurons transfected with Syn-GFP and the indicated RNAi using Lipofectamine 2000 (<1% transfection efficiency). Neurons were loaded with FM 4-64, and three images were captured every 30 s to confirm that the positive FM 4-64 sites were stationary presynaptic terminals. Arrows indicate FM 4-64–positive sites on transfected axons. FM dyes were then unloaded to demonstrate specificity (A′–F′). (F′) The FM 4-64–positive site (arrowhead) not observed in dye-“load” image (E), but observed following dye-“unload” (E′) most likely represents a mobile FM 4-64–positive puncta on an untransfected neuron. The FM 4-64 cluster in the transfected neurons (arrow) is not observed after de-staining. The density (G) and size (H) of FM 4-64-positive puncta ± SE were reduced in cells expressing RNAi-3 compared with control. N = 17 cells and >85 FM-4-64 puncta from more than three separate cultures. *p < 0.05 using Student’s t test. Scale bar, 5 μm.

使用方法

  1. 储存液配制

于实验前，将冻干粉置于室温回温至少20min，加入无菌水或DMSO配制成10mM或其他浓度储存液，比如，对于1mg FM 4-64（Mw: 607.51）加入164μl DMSO,充分溶解后即得到10mM储存液，根据单次用量分装，≤-20℃冻存，避免反复冻融。

  1. 染色方法（仅作参考）

【注意】：

a）以下以爬片生长的贴壁活细胞的质膜染色为例，仅作参考。最佳的染色条件根据使用细胞特征进行调整。

b）由于FM 4-64快速被内吞，很有必要参考下方的温度和时间指导来减慢内吞，提高质膜的选择性标记和成像。内吞很可能在染色的10min内发生。

c）以下步骤推荐使用不含钙镁的HBSS（MS3505-500ML）。钙镁的存在明显会加速染料内吞，导致质膜选择性染色很弱。

2.1用提前冰浴的HBSS缓冲液来稀释储存液到所需的工作浓度比如8μM。

2.2 将盖玻片从培养基内取出，快速的浸入含足量FM 4-64的染色工作液，冰上孵育1min。质膜能被快速染色。

2.3 将盖玻片从染色工作液中取出，置于载玻片上封片，周围用石蜡密封，置于冰上，立即成像。

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