形成、限制与观察基于微管的活性向列相

摘要

本文介绍了由微管和驱动蛋白马达制备活性向列相的方法，包括蛋白质的制备与构建，以及利用微井实现活性向列相的限制。

摘要

基于生物大分子的活性相的形成已成为研究人员探索活性液晶这一新兴领域及其在细胞生物学中可能作用的重要技术。这些新型系统由能够局部消耗能量的自驱动亚单元组成，从而产生非平衡态的动态流体。为了形成本文所述的活性液晶相，需将包括生物大分子和分子马达在内的纯化蛋白质组分混合，并在三磷酸腺苷（ATP）存在下自发形成活性向列相。为了观察向列态，必须将材料限制在适合显微镜观察的几何结构中，并达到足够高的密度。本文介绍了两种利用微管和驱动蛋白马达构建活性向列相的方法：在油水界面处组装二维活性层，以及在弹性体微腔中利用油层覆盖进行组装。同时还描述了将活性材料置入不同形状小型微腔的技术。

引言

活性流体由从局部环境中获取能量的粒子或组分构成，这些粒子或组分能够利用能量驱动自身运动。在适当条件下，这些具有运动能力的活性组分可协同作用，在大尺度上产生涌现性的流体动力学行为。文献中已报道了多种此类非平衡态相行为的例子，活性相广泛存在于各类生命系统中。一些典型实例包括细菌菌落¹、细胞层^2,3，以及生物体的成群运动或集群行为^4,5。活性相也在细胞骨架丝状结构的凝聚相中得到了广泛研究，无论是在细胞内部⁶，还是在利用生物提取组分构建的合成系统中均有涉及^7,8,9。天然或合成生物提取系统中形成的液晶有序结构及其拓扑缺陷的产生尤其受到研究领域的关注。近年来，多个研究团队已对这些系统、其基本物理特性及其在生物学中的意义进行了深入探讨^2,3,10,11。

本文重点研究由微管和驱动蛋白马达蛋白组合形成的活性向列相态。传统的向列相液晶是一种处于热力学平衡态的物质相，其组成分子表现出取向有序性。例如，由相对刚性的棒状分子组成的流体可能在较低温度下呈现向列相，而在较高温度下则转变为无取向的各向同性流体相。¹²首个活性向列相的实验实例由 Sanchez 等人开发。¹³，改编自早期方法 体外 实验¹⁴ 其中利用运动蛋白的簇集在相邻微管束之间产生剪切运动。当该微管系统被限制在薄层中时，会自发出现向列相有序结构。近年来，多个实验团队已对活性向列相态进行了深入研究^15,16 和理论的^17,18 研究团队，聚焦于主动湍流等现象——即流体产生自驱动混沌流动的状态¹⁹ —以及可移动的拓扑缺陷。本文描述了利用微管和驱动蛋白马达在不同实验几何条件下制备和形成活性向列相的方法。首先介绍各组分溶液的制备方法，随后介绍使用两种不同流动腔几何结构形成活性向列相的实验步骤。展示了典型的成像结果。最后，描述了将活性向列相限制在微孔和微通道中的方法。

方案

1. Preparing the active material

NOTE: The 2D active nematic is assembled in a three-step process. First, two separate solutions are prepared: a) polymerized, stabilized microtubules and b) MIX (a solution containing kinesin motors). These are combined and activity is initiated upon adding adenosine triphosphate (ATP). The material is then confined in a suitable geometry, such that its density is high enough for nematic order to emerge. Protocols are included for the preparation of all necessary components and how to assemble the active phase.

1. Kinesin motor protein cluster preparation
   1. Express and purify recombinant K401-BIO motor proteins (K401 motors) from Escherichia coli following the protocol by Edgar C. Young²⁰.
      NOTE: K401-BIO motor proteins are dimeric and consist of two heads connected with a helical stalk. The motors were supplied by the Brandeis Biomaterials Facility and used as previously reported¹⁶. For the purposes of forming an active nematic, the kinesin molecules are biotinylated and then connected via a streptavidin linkage to form kinesin clusters of up to four motors^13,15,21,22. It is helpful to express the kinesin with a green fluorescent protein (GFP) tag.
   2. After purification and incubation of streptavidin and motors on ice for 40 min, flash-freeze the K401 motors in liquid nitrogen in 5 µL aliquots at a final concentration of 0.7 mg/mL, and store at -80 °C.
      NOTE: The experiment can be paused here. Gently thaw the kinesin when needed and do not refreeze.
   3. Prepare clusters of biotin-labeled kinesin (KSA) by mixing 24 vol% of 0.7 mg/mL K401 motors, 27 vol% of 0.325 mg/mL streptavidin, and 3 vol% of 5 mM dithiothreitol (DTT) (to prevent aggregation) at 4 °C in 46 vol% M2B buffer (80 mM PIPES [1,4-Piperazinediethanesulfonic acid, pH 6.8], 2 mM MgCl₂, and 1 mM EGTA [ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid]). Let the KSA incubate on ice for 40 min.
2. Microtubule solution preparation
   NOTE: Guanosine-5'-[(α,β)-methyleno]triphosphate, sodium salt (GMPCPP) is a slowly hydrolysable analog of Guanosine triphosphate (GTP), and microtubules formed in the presence of GMPCPP are three times stiffer than GTP microtubules²³ and shorter. The use of short, stiff microtubules is favorable for the formation of the active nematic phase as these factors combine to promote liquid crystalline ordering.
   1. Polymerize unlabeled cycled tubulin (99%, see Table of Materials) using 0.6 mM GMPCPP at a tubulin concentration of 6 mg/mL.
      NOTE: High quality tubulin can be also purified from bovine or porcine brain following established protocols or obtained from another reliable source such as the Brandeis Biomaterials Facility where materials can be shipped frozen to prevent damage. For tubulin purification from bovine brain, please refer to the published protocol from Bate et al.²⁴. For tubulin purification from porcine brain, refer to the published protocols from Castoldi et al.²⁵ and Tayar et al.²⁶.
   2. In preparation for polymerization, prepare a heat bath at 37 °C and pre-cool the centrifuge to 4 °C. Combine the unlabeled tubulin solution in M2B buffer (step 1.1.3) in a 500 µL ultracentrifuge tube with 4 mol% rhodamine-labeled tubulin (see Table of Materials) to produce 4% labeled microtubules after polymerization.
   3. Verify tubulin concentration using a Bradford assay²⁷. The total tubulin concentration in the centrifuge tube should be 6.5-6.9 mg/mL.
   4. Incubate the tubulin mixture on ice for 10 min and ultracentrifuge for 10 min at 352,700 x g at 4 °C. This step removes dysfunctional tubulin, which will be in the pellet.
   5. Using a pipette, carefully extract the supernatant containing functional tubulin into a microcentrifuge tube. Add GMPCPP to a final concentration of 0.6 mM to induce tubulin polymerization and DTT to a final concentration of 1 mM to prevent protein aggregation.
   6. Incubate the mixture in the heat bath at 37 °C for 30 min, then centrifuge again for 10 min at 14,000 x g at room temperature. Remove the supernatant, then dilute the pellet with M2B buffer to reach a final microtubule concentration of 6 mg/mL.
      NOTE: This solution can be stored at room temperature for at least 4 h before use.
   7. To check that the microtubules have polymerized successfully, dilute 1 µL of the microtubule solution 100:1 with M2B buffer and pipette on a microscope slide. Cover with a cover slip for imaging using a fluorescence microscope (see Table of Materials) with a 40x objective lens.
      NOTE: Excitation and emission wavelengths are chosen according to the fluorescence labeling used on the microtubules. In this protocol, rhodamine labeling is used (see step 1.2.2), so imaging is carried out using an excitation band of 515-560 nm and a 590 nm long pass filter. Figure 1 shows a representative example.
   8. After polymerization is complete, drop-freeze the microtubules as 2 µL aliquots in liquid nitrogen and store at -80 °C (if required).
3. Preparation of MIX
   NOTE: MIX is an aqueous solution that includes the kinesin-streptavidin clusters (KSA). Prepared MIX should be stored at -80 °C in 4 µL aliquots prior to the experiment. When MIX is combined with ATP and the microtubule solution described in step 1.2 at room temperature, activity is initiated. MIX is prepared as follows^13,19.
   1. Prepare a solution for preventing fluorescence fading (ANTIFADE) by mixing two antioxidant solutions. Combine AO1 (250 mM DTT, 65 mM catalase) and AO2 (750 µM catalase, 3 mM glucose oxidase) in a 1:1 volume ratio. Include 20 mM Trolox, another antioxidant used to reduce damage caused by fluorescence microscopy.
   2. Prepare MIX by making a solution of KSA (described in step 1.1.3) that includes 6 wt% 20 kDa polyethylene glycol (PEG) to induce bundling of the microtubules, 3 vol% ANTIFADE, and 5 vol% pyruvate kinase/lactic dehydrogenase (PKLDH) at 70 mg/mL for ATP regeneration.

2. Creating the active nematic

NOTE: Activity in the material is initiated by ATP addition. The active network is prepared fresh for each experiment by adding ATP at a concentration high enough to induce motor activity. To form a uniform, fully developed active nematic phase, the microtubules must be at a sufficiently high density. This can be achieved by confining the microtubules in between two immiscible fluids to form a two-dimensional (2D) active nematic layer. This method was originally developed at Brandeis University¹³ and remains a popular technique for producing a homogeneous, high quality, active nematic phase.

1. Flow cell method for active nematic formation
   1. Prepare hydrophilic coverslips with an acrylamide coating.
      1. Clean the coverslips thoroughly with soapy water, ethanol, and 0.1 M NaOH with alternate rinses using nanopure water. Once rinsed, coat the coverslips with a silane solution composed of 100 mL of ethanol, 1 mL of acetic acid, and 500 µL of 3-(trimethoxysilyl) propylmethacrylate for 15 min, then rinse with nanopure water.
      2. Prepare an acrylamide solution from 95 mL of nanopure water and 5 mL of 40 wt% acrylamide, then degas the solution for 30 min in a vacuum oven.
      3. Add 35 μL of tetramethylethylenediamine (TEMED) for a 2.3 mM final concentration and then 0.07 g of ammonium persulfate. Pour the acrylamide solution over the coverslips while face up and incubate overnight at room temperature.
   2. Prepare hydrophobic microscope slides. Pipette 100 µL of a water repellent solution (see Table of Materials) onto a clean glass microscope slide, then place another clean glass slide on top. This ensures an even coating of the water repellent solution on the surface where it sits for 2 min. After 2 min, remove the second glass slide and rinse the first slide thoroughly with nano-pure water, then dry with nitrogen gas. Gently clean the region where the tape will be placed (around the pattern) with acetone to ensure the water repellent solution does not prevent adhesion to the glass.
   3. Prepare a mixture of engineered oil that includes 1.8% (v/v) 008-FluoroSurfactant (see Table of Materials).
   4. Assemble the glass slide and coverslip with the hydrophobic glass slide on the bottom of the flow cell and the hydrophilic cover slip as the upper surface in a sandwich geometry using 40 µm double-sided adhesive spacers. Place the spacers 1.5 mm apart on the hydrophobic microscope slide. Then place the acrylamide coated coverslip on top of the spacers with the treated side face down to adhere. Ensure the adhesion is complete with pressure from a blunt object.
      NOTE: The goal is to assemble a flow cell with a flat oil/water interface inside (Figure 2A,B). This is where the active layer will form. Alternative spacer tapes and films can also be used to produce a similar cell thickness.
   5. After the flow cell has been constructed, immediately pipette the oil mixture into the flow cell, filling the enclosed space.
   6. Using a pipette, in a separate vial gently mix 6 µL of active material with 3.73 µL of MIX, 1 µL of microtubule solution, 0.6 µL of ATP solution (the concentration can be varied to vary microtubule velocity), and 0.67 µL of M2B buffer.
   7. Pipette freshly mixed active material into one open end of the flow cell, volumes will vary but should exceed the volume of the flow cell (roughly 3-6 μL). Some oil will be displaced by the aqueous solution as it is injected into the channel; this can be wicked up at the opposite end of the flow channel using a small piece of tissue paper.
   8. After filling, seal both sides of the flow cell with an epoxy glue (see Table of Materials) that hardens when exposed to UV light for 20 s.
      NOTE: At this point, the active material forms a 3D network that remains suspended in the water layer.
   9. To confine the active layer between the two immiscible fluids in a quasi-2D layer, place the flow cell in a swinging bucket centrifuge (see Table of Materials) with the aqueous phase on top and the denser oil layer underneath. Centrifuge at 212 x g for 10 min. After this step is complete, the flow cell can be taken to an epi-fluorescence microscope for imaging with a 10x or 20x magnification objective. Figure 2C,D shows typical images before and after this step.
2. Inverted method for active nematic formation
   NOTE: An alternate method to that described in step 2.1 is to assemble the active nematic layer underneath a thick oil layer confined to a deep PDMS well²⁸. This method produces similar results, and is somewhat easier to master; however, image quality using this method is usually not as good as the flow cell method.
   1. Prepare the polydimethylsiloxane (PDMS) using an elastomer curing agent and an elastomer base (see Table of Materials). Mix the two components in a 1:10 ratio using a metal spatula. Tiny bubbles appear during mixing which are difficult to remove, and the mixture appears milky. To remove these bubbles, place the mixture under a vacuum to degas for 1 h, after which the uncured PDMS should appear transparent.
      NOTE: The PDMS is now ready to form any shape using a mold, or it can cure in the container and then be cut or punched in the desired pattern.
   2. Pour the PDMS into a suitable mold and leave overnight to cure at 60 °C.
      NOTE: Untreated, the surface of cured PDMS is hydrophobic, but with surface treatment, it can be made hydrophilic.
   3. To prepare a hydrophilic PDMS surface, coat the PDMS with an acrylamide polymer brush²⁹. This step also prevents proteins from sticking to the surface.
      1. Start by cleaning the PDMS for 10 min with both ethanol and isopropanol, then rinse thoroughly with deionized water 3x and dry. Use a plasma cleaner for 5 min to clean the dry, cured PDMS. This step makes the surface more hydrophilic.
      2. Next, prepare a silane solution (98.5 wt% ethanol, 1 wt% acetic acid, and 0.5 wt% Trimethoxysilyl propyl methacrylate) and immerse the substrate in that solution for 15 min to prepare for the acrylamide coating. Rinse the substrate thoroughly with deionized water and immerse in acrylamide solution (2 wt% acrylamide/bis solution, 2.3 mM TEMED, and 3 mM ammonium persulfate).
         NOTE: These substrates can be stored at room temperature covered in acrylamide solution in a glass Petri dish, and should be used within 2 weeks.
   4. When ready for use, rinse the surface with deionized water and dry with nitrogen for immediate use. Add the active mixture (described in step 2.1.6) to the wells and immediately add silicone oil on top to a thickness of approximately 2 mm.
      NOTE: At this stage, the active mixture will be sandwiched between the oil and the hydrophilic coating on the PDMS, but it will still be somewhat three-dimensional.
   5. To push the material further into a 2D layer, glue the PDMS device onto a glass slide, place it in a swinging bucket centrifuge and spin for 12 min at 212 x g. The device will need to be positioned such that the silicone oil is on the top of the aqueous layer. Representative results are shown in Figure 3.

3. Preparing active nematics in confined geometries

NOTE: Active nematics such as this quasi-two-dimensional system can be challenging to confine into small microfluidic geometries such as wells or channels. Here, a reliable method to confine the material into different shaped PDMS wells is described.

1. First, design a master mold for the PDMS. This can be achieved by 3D printing pillars on a substrate. After 3D printing the resin master mold, clean with isopropanol and then cure the mold under a UV lamp for 45 min and in the oven at 120 °C for 2 h (Figure 4).
   NOTE: Curing under UV and thermal post-curing improves the quality of replication in PDMS by removing monomers and photo inhibitor residues from the resin³⁰.
2. Use the master mold to create wells from PDMS. Prepare the PDMS as described in step 2.2.1. Immerse the master mold in uncured PDMS and cure overnight in an oven at 60 °C.
3. After PDMS curing is complete, carefully remove the master mold and cut the PDMS as desired to work with the wells (Figure 4). Treat the PDMS surface as described in step 2.2.3. Before the experiment, the surface can be attached to a glass slide with epoxy glue to make imaging easier.
4. Pipette 1 µL of the active mixture described in step 2.1.6 onto the PDMS substrate and immediately add silicone oil with 100-1000 cSt viscosity²⁸ on top of the active network droplet. The active network will move into the well; this process takes up to 60 min (Figure 4). As described in step 2.2.5, the 2D network can be enhanced by spinning down the PDMS well in the swinging bucket centrifuge for 12 min at 212 x g.

结果

图1展示了由GMPCPP微管蛋白制备的单根微管的代表性图像。图像中显示了长度相近的短微管（存在一定分散性）。对微管溶液进行充分稀释后，应能获得彼此分离良好的微管图像，以便验证其长度。由于微管尺寸较小，单独成像可能存在挑战。建议使用专为荧光显微镜设计的高灵敏度相机以获得最佳效果。图2和图3分别展示了采用流动池法（第2.1节）和倒置法（第2.2节）成功实验所获得的荧光显微图像示例。良好形成的活性向列相层在纹理上均匀一致，无明显空洞区域，并存在可移动的拓扑缺陷。但需注意，缺陷核心处可能存在一些可接受的小型空隙。除图2和图3所示示例外，还提供了三个补充视频（视频1、视频2和视频3），用于展示成功实验中活性向列相应呈现的形态。所有视频均显示活性向列相具有平滑连续的运动特征。材料达到稳态后，微管浓度无明显变化。只要系统中存在足够的ATP，材料将持续保持均匀运动。

图1：GMPCPP微管的荧光显微镜图像。 GMPCPP微管以4%罗丹明标记的微管蛋白进行标记，并在37 °C下聚合20分钟。成像在室温下进行。比例尺 = 10 µm。 请点击此处查看该图的放大版本。

图 2：流动池中的微管向列相。 （A）流动池的横截面示意图，几何尺寸为 1 mm × 18 mm。（B）流动池的俯视示意图。（C）荧光显微镜图像，显示活性溶液在油/水界面组装前的典型外观。（D）活性向列相在流动池内油/水界面处组装后的荧光显微镜图像。比例尺 = 100 µm。 请点击此处查看该图的放大版本。

图 3：采用倒置法制备的活性向列相的荧光显微镜图像。 比例尺 = 200 µm。请点击此处查看该图的放大版本。

图4：流程图，展示在PDMS微腔中实现活性向列相材料限制的方法，包括模具制备和表面处理。 右侧图像（受限的活性材料）中的比例尺 = 200 µm。请点击此处查看该图的放大版本。

视频 1：使用流动池法制备的活性向列相的代表性结果。 请点击此处下载该视频。

视频 2：采用倒置法制备的活性向列相的代表性结果。 请点击此处下载该视频。

视频3：使用倒置法制备并限制在椭圆形微井中的活性向列相的代表性结果。 请点击此处下载该视频。

讨论

在整个实验方案中，实验人员有几个关键步骤可以进行重要检查。在将任一装置填充活性材料之前，应使用荧光显微镜（见图1）检查微管是否已聚合，理想长度约为2–3 µm。若在显微镜下无法观察到微管，则可能已发生去聚合，导致活性向列相无法形成。由于单个微管非常细小，直接通过显微镜观察可能存在困难。本研究中使用了专为低光照条件设计的高质量荧光相机，并结合配套软件来确认丝状结构的生长情况。此阶段不应出现明显的荧光聚集体，否则可能提示去聚合或存在变性蛋白。此外，建议制备一个简单的显微镜检测载片，将微管、MIX和ATP按方案中所述比例混合。组分混合后应立即出现活性，材料状态应与图2C所示相似，可见束状结构及明显的丝状物运动。

使用流动池方法时，离心时间和流动池的取向对于形成均匀的活性层至关重要。此步骤可能需要根据所使用的离心机类型进行一些细微调整。将流动池的活性面垂直于旋转平面进行离心，可获得最佳效果，因为这样可使材料均匀地推向液体界面。离心前务必仔细检查流动池是否已密封良好。

采用倒置法制备受限活性向列相时，有几个步骤需要优化。首先，必须使用能够产生高分辨率结构的3D打印方法。不平整的侧壁可能导致微管滞留，从而扰乱流动。微孔不宜过深（本研究中使用的微孔深度为150-200 µm，并覆盖2 mm厚的油层）。实验人员可能需要通过反复试验略微调整这些参数，以获得最佳结果。

不同作者已采用流动池法和倒置法研究了多种影响活性流动的因素，包括不同类型的油¹²和浸没结构¹³。方法的选择取决于实验目的。由于覆盖液体的不同，流动池法在活性层上方进行光学成像时，成像清晰度优于倒置法。在流动池法中，成像是透过玻璃盖玻片和一层薄水进行的，而倒置法则将油层置于上方。这意味着倒置法需要使用长工作距离物镜，且成像质量有所降低。通过比较图2D（流动池法）与图3（倒置方法），以及分别对应的视频 1和视频 2，可以观察到成像质量的差异 。图3所用物镜的放大倍数较低，但工作距离更长，而图2所用物镜则相反。如果具备合适的倒置显微镜，并搭配适用于载玻片基底且工作距离适当的物镜，则可避免倒置法的这些成像劣势。此外，可使用更薄的玻璃作为基底，以允许使用标准工作距离的物镜。

其优势在于，倒置构型可使用更广泛黏度范围的油，且不一定需要摆动桶式离心（如果设备不可用），并且一旦模具制备完成，系统的组装相对更简便。然而，采用倒置法在孔中进行限域时，可能需要一定的离心步骤，以使材料形成明确的二维层状结构。

近年来，流动池法在需要连续活性层的实验中取得了非常成功的应用。我们近期的研究关注了活性层中拓扑缺陷的动力学行为，其中高质量成像和纹理分析至关重要¹⁹。此外，流动池法还被用于研究油浸微结构对活性流动的影响¹⁶，以及利用柱状结构捕获活性流动中的缺陷³¹。该方法在形成连续活性层方面效果良好，且成像质量优异。然而，用于制备最终二维活性层的离心步骤操作较为困难，且流动池容易发生泄漏和产生气泡。倒置法是一种非常有用的替代方案，成功率高，易于构建，只要能够制备出高分辨率的三维打印主模具，即可适用于任意基底图案或几何形状。该方法在研究几何限域对活性向列相动力学影响方面也具有优势，因为它使填充微井的过程相对简便。

本文介绍了两种由微管和驱动蛋白马达构建活性向列相的方法，以及一种将材料限制在微孔中的技术。该系统是目前文献中最为清晰的活性向列相实例，已被全球多个研究小组成功复现。这种材料的重要性不仅在于其组分具有生物学来源，更在于它开辟了活性有序流体研究的全新方向。通过研究该系统并阐明其基本特性，科学家有望推进全合成活性相的设计与实现。

聚焦于限制条件对活性向列相影响的实验，有望回答关于拓扑限制下活性流动和拓扑缺陷动力学行为的基本问题。本文介绍的方法将有助于开展多种以几何结构为核心的实验及其分析，包括微流控和活性混合研究。

材料

本文使用的材料清单

姓名	公司	目录编号	评论
20 kD PEG（聚乙二醇））	Sigma Aldrich	1419109	消耗剂 CAS 号：125061-88-3
3-（三甲氧基硅烷基）甲基丙烯酸丙酯	Sigma Aldrich	M6514-50ML	CAS 号：2530-85-0
3D 打印机 &树脂	Phrozen		Phrozen 声波迷你 8K 3D 打印机 - 水灰色 8K 树脂
40% 丙烯酰胺溶液	BIO-RAD	1610140	CAS 编号：7732-18-5、79-06-1
乙酸	Fisher		CAS 编号：64-19-7
丙酮	Sigma Aldrich		CAS 编号：67-64-1
粘合片材（注意："石蜡膜"是一种替代品）	Grace Bio-Labs	620001	SecureSeal
过硫酸铵	Sigma Aldrich	A3678	CAS号：7727-54-0
Aquapel （注："RainX"是一种替代品）	Aquapel 玻璃处理		水性玻璃处理
ATP（三磷酸腺苷）	Sigma Aldrich	A1852	CAS号：34369-07-8
烧杯	VWR
过氧化氢酶	Sigma Aldrich	C9322	CAS 编号："9001-05-2"
干燥器	Bel-art
数码 CMOS 相机	滨松	ORCA - Flash4.0 LT+
DTT（二硫苏糖醇）	Sigma Aldrich	D9779	CAS 编号："3483-12-3"
EGTA（3,12-双（羧甲基）-6,9-二恶杂-3,12-二氮杂四癸烷-1,14-二酸）	Sigma Aldrich	MFCD00004291	CAS 号：67-42-5
乙醇	Sigma Aldrich		CAS 号：64-17-5
荧光显微镜	Leica	DM 2500P
玻璃盖玻片	VWR	48368-040
载玻片	VWR	16004-430
葡萄糖	Sigma Aldrich	G7021	CAS 号：50-99-7
葡萄糖氧化酶	Sigma Aldrich	345386	CAS号：9001-37-0
GMPCPP（鸟苷酸酯 5'-&α;，&β;-亚甲二膦酸盐）	Jena Bioscience	NU-405S	CAS号：14997-54-7
HFE7500油	3M
热板	Fisher Scientific	添益隔热板型号 100M
异丙醇	VWR
KCl（氯化钾）	Sigma Aldrich	P5405	CAS号：7447-40-7
甲醇	Sigma Aldrich		CAS号：67-56-1
MgCl2 （氯化镁）	Sigma Aldrich	208337	CAS号：7786-30-3
微量离心管	Eppendorf - Thermo Fisher	1.5 mL
纳米纯水器	Sartorius	arium mini
NaOH（氢氧化钠）	Sigma Aldrich	SX0603	CAS号：1310-73-2
培养皿	VWR
PH 计	Thermo Scientist	Orion 3 STAR
磷酸烯醇-丙酮酸 （PEP）	Sigma Aldrich	MFCD00044476	CAS号：4265-07-0
PIPES （1,4-哌嗪二烷磺酸）	Sigma Aldrich		CAS 编号：5625-37-6
移液器（0.2 - 1000 µl）	VWR
Pluronic F-127	Sigma Aldrich	2594628
RAN 表面活性剂（注：Emulso 的"FluoSurf"是一种替代品）	Ran Biotechnologies	008-氟表面活性剂-2wtH-50G
硅油（100mpa s-1000 mpa s）	Sigma Aldrich		CAS 编号：63148-52-7
链霉亲和素	Thermofisher	S888
水平吊篮离心机	Thermo Scientist	Sorvall legend RT+
Sylgard 184 弹性体底座	World Precision Instruments	SYLG184
Sylgard 184 弹性体固化剂	World Precision Instruments	SYLG184
台式离心机	Eppendorf	MiniSpin Plus
TEMED （四甲基乙二胺）	BIO-RAD	1610800	CAS 编号：110-18-9
Trolox（6-羟基-2,5,7,8-四甲基色-2-羧酸）	Sigma Aldrich		MFCD00006846 CAS 编号：53188-07-1
微管蛋白	细胞骨架	T240-B
微管蛋白（罗丹明标记）	细胞骨架	TL590M-A
超速离心机	Beckman	Optima Max-TL
UV Light	RapidFix
UV 固化胶（注意："Norland NO81"是一种替代品）	RapidFix
水浴	Thelco
Whatman 滤纸	Sigma Aldrich	WHA1001325