FM 4-64FX (Fixable analog of FM 4-64) Synaptic vesicle 突触小泡

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 4-64FX (Fixable analog of FM 4-64)

 MX4024-100UG 100μg

FM 4-64FX (Fixable analog of FM 4-64) MX4024-200UG 2×100μg

产品描述

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烈的长波长红色荧光。FM 4-64与呈绿色荧光的FM 1-43（MX4014）在合适的滤片下能够区分开，允许双色实时观察膜的再循环。

FM 4-64FX是可固定的FM 4-64（MX4016）类似物，能承受基于醛类的固定剂（比如：多聚甲醛或戊二醛）。

产品特性

1）分子式：C35H45N4O6F9

2） 分子量：788.75

3） 纯度：≥90%（HPLC）

4） 外观：固体 

5） 溶解性：溶于水、DMSO

6） Ex/Em：565/744nm（in MeOH）（见附录I）

7） 化学结构式：

保存与运输方法

保存：2-8°C避光干燥保存，或置于-20℃避光干燥长期保存，至少1年有效。 

运输：室温运输。

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1） Ayala-Lujan JL, Ruiz-Perez F. Adhesion of Enteroaggregative E. coli Strains to HEK293 Cells. Bio Protoc. 2018 Apr 20;8(8):e2802. doi: 10.21769/BioProtoc.2802. PMID: 34286021; PMCID: PMC8275309.

使用方法（EAEC binding assay in HEK293 cell suspension）：

Binding assays are performed with cell suspensions in 15 ml conical tubes as follows:

  1. Stain approximately a total of2 x 106 cells in suspension by adding 300 µl of 5 µg/ml of FM 4-64FX and 4’,6’-diamidino-2-phenylindole (DAPI) for nucleus visualization for 5 min, followed by incubation with EAEC expressing GFP.

Note: DAPI can also be added at the end of the experiment as part of the anti-fade solution. HEK293 cell membranes are stained with FM 4-64FX before EAEC infection to avoid staining of bacterial membranes. If wish, FM 4-64FX staining can be skipped and proceed with the infection with fluorescent bacteria.

  1. Remove the excess of FM 4-64FX dye on cells by adding 10 ml of HBSS to the tubes and spinning cells down at 250 × g(~1,500 rpm) for 5 min.
  2. Mix 1 x 106FM 4-64FX-stained HEK293 cell derivatives expressing or not Muc1 with approximately 2 x 107 CFUs of GFP-EAEC strains to a multiplicity of infection (MOI) of 1:20 (cell:bacteria) in 1 ml of fresh DMEM medium. Incubate the cells for 1 h at 37 °C and 5% CO2.
  3. After 1 h incubation, wash the cells three times by adding 10 ml of HBSS buffer and by gently inverting the tube 2-3 times. Centrifuge the cells at 100 × g(~1,000 rpm) for 2 min to allow cell precipitation, but minimizing unbound bacteria sedimentation. Repeat HBSS-washes two more times.
  4. Remove the supernatant by aspiration using a disposable transfer pipette or a vacuum system (care should be taken not to disturb or aspirate the cells at the bottom of the tube; most unbound bacteria settle at later times and lower speeds).
  5. Fix cells by resuspending in 100 µl of PBS-5% formalin (g., 50 µl PBS + 50 formalin at 5% [v:v]). Examine the cells immediately or save at 4 °C to analyze it later, preferably during the first 48 h.
  6. Use 10 µl of cell suspension to prepare a smear on glass slides by extending the cell suspension with a coverslip. Let smear to dry out for about 10 min, then coat smear with a drop of anti-fade-DAPI.
  7. Analyze the slides by using a fluorescence microscope equipped with wavelength filters for blue (365/10 excitation/420LP emission), green (480/20 excitation/510LP emission), and red (535/30 excitation/580LP emission). You will see DNA stained in blue, cells membranes stained in red and bacteria in fluorescent green. A representative example of a binding assay with the prototype EAEC 042 strain and its isogenic fimbria mutant (042aafA) is illustrated in Figure 1.

Fig 1. Binding of EAEC to HEK293-Muc1 cells. B. HEK293 and HEK293-Muc1 cells were infected in suspension with EAEC strain 042 or with its isogenic fimbria mutant (042aafA), each expressing a GFP reporter and analyzed by fluorescence microscopy (40x). EAEC042 is in green, cell membranes are stained in red with FM 4-64FX, and DNA is stained in blue with DAPI.

文献2）Corrales RM, Vaselek S, Neish R, Berry L, Brunet CD, Crobu L, Kuk N, Mateos-Langerak J, Robinson DR, Volf P, Mottram JC, Sterkers Y, Bastien P. The kinesin of the flagellum attachment zone in Leishmania is required for cell morphogenesis, cell division and virulence in the mammalian host. PLoS Pathog. 2021 Jun 18;17(6):e1009666. doi: 10.1371/journal.ppat.1009666. PMID: 34143858; PMCID: PMC8244899.

使用方法（Uptake of FM4-64FX）：

Microscopic analysis of FM4-64FX uptake was carried out as previously described [5] with slight modifications. Briefly, exponentially growing promastigotes (5×106 cells) were incubated 10 min in the dark at 27°C in complete HOMEM medium with 10μg/mL FM4-64FX. After incubation, 1 mL of cold PBS was added to stop up-take. Cells were fixed with cold 2% paraformaldehyde solution in PBS at 4°C during 5 min. Cells were resuspended in 100 μL PBS with 10μg/mL Hoechst 33342 and imaged immediately on poly-lysine coated slide.

Fig 2. Deletion of FAZ7B results in delayed incorporation of the membrane marker FM4-64FX into the flagellar pocket and altered flagellar pocket segregation. (A) Microphotographs representative of the flagellar pocket labelling with FM4-64FX (red) in the parental and FAZ7B KO cell lines. DNA was stained with DAPI (blue). Scale bar: 5 μm. (B) Quantification of FM4-64FX labelling in the flagellar pocket in 1N1K parental and FAZ7B KO cell lines; mean values ± SD of three independent experiments. ** p < 0.01 (Student’s t-test).

文献三、Zhang F, Sun Y, Pei W, Jain A, Sun R, Cao Y, Wu X, Jiang T, Zhang L, Fan X, Chen A, Shen Q, Xu G, Sun S. Involvement of OsPht1;4 in phosphate acquisition and mobilization facilitates embryo development in rice. Plant J. 2015 May;82(4):556-69. doi: 10.1111/tpj.12804. PMID: 25702710.

使用方法（plasma membrane localization）：Isolation of rice protoplast from culms of etiolated seedlings and subsequent transfection were performed as described previously. For plasma membrane localization, a 5 μg ml−1 solution of the membrane-selective fluorescent vital dye FM4-64FX was used to stain the transfected protoplasts. Protoplasts were observed under a 60× objective. The fluorescence of FM4-64FX and GFP in cells was analyzed using a 543 nm helium/neon laser and a 488 nm argon laser, respectively, using an LSM410 confocal laser scanning microscope.

Fig 3. Subcellular localization of OsPT4. Confocal laser scanning microscopy images of rice protoplasts transiently expressing 35S::EGFP (left panels) or 35S:: OsPT4:: EGFP (right panels). Scale bars = 5 μm.(a) Bright-field images.(b) Confocal images of protoplasts under GFP channel only.(c) FM4-64FX dye-induced red fluorescence showing the position of the plasma membrane.(d) Merged images of GFP fluorescence (green) and FM4-64FX fluorescence (red).

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5mM或其他浓度储存液，比如，对于100μg FM 4-64FX（Mw: 788.75）加入25.3μl DMSO，充分溶解后即得到5mM储存液，根据单次用量分装。需注意，FM 4-64FX的储存液相对于FM4-64来说不稳定，请置于≤-20℃冻存，于2周内使用，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。

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