Danyang Li and Khuloud T. Al-Jamal
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Owing to the unique physical and chemical properties of carbon nanotubes, they have been widely explored as delivery vectors for proteins, and nucleic acid etc. after functionalization. Particularly, the modification of carbon nanotubes suited for the delivery of siRNA has been intensely studied over the past decade. The assay described in this chapter allows for realizable quantification of siRNA binding on carbon nanotube-based materials using gel electrophoresis and silencing by flow cytometry when the siRNA complexes are delivered in vitro.
由于碳纳米管独特的物理和化学性质，它们在经过功能化后被广泛研究作为蛋白质和核酸等的递送载体。特别是，适用于 siRNA 递送的碳纳米管修饰在过去十年中得到了深入研究。本章描述的检测方法通过凝胶电泳对碳纳米管基材料上 siRNA 的结合进行可行的定量，同时在体外递送 siRNA 复合物时，通过流式细胞术实现其沉默效果。
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siRNAs are short stretch dsRNAs, which degrade the complementary mRNA in the cytoplasm. When delivered into cytoplasm, siRNAs are loaded to RNA-induced silencing complex (RISC), which contains Argonaute 2 (Ago-2) that cleaves and releases one strand from the dsRNA. The resulting single-strand RNA (guide siRNA) would direct the targeted mRNA through complementary base pairing. The targeted mRNA is cleaved by Ago-2 into small pieces, therefore causing mRNA degradation and gene silencing.
siRNA 是一种短链双链 RNA，能够在细胞质中降解互补的 mRNA。当 siRNA 进入细胞质后，会被加载到 RNA 诱导沉默复合物（RISC）中，其中含有 Argonaute 2（Ago-2），负责切割并释放双链 RNA 的一条链。生成的单链 RNA（引导 siRNA）会通过互补碱基配对来引导目标 mRNA 的降解，从而实现基因沉默。
Making use of the unique features of siRNA, several types of siRNA delivery systems such as viral and nonviral delivery systems, which include cationic polymers, lipids, nanoparticles, electroporation, microinjection, biolistics, polypeptide, and extracellular vesicle, have been developed in the past decades to efficiently transport siRNAs to the desired destination with minimal adverse effects. The interest in designing nonviral vectors for gene delivery has never waned as they are less hazardous in terms of antigen-specific immune responses compared to viral vectors, although in general transfection efficiency of nonviral vectors are lower. Encapsulation of naked RNA by cationic polymers or lipids such as Lipofectamine® 2000 have been a major player in commercial products although they present some cytotoxicity and short-term transfection .
基于 siRNA 的独特特性，过去几十年间开发了多种 siRNA 递送系统，包括病毒和非病毒载体，如阳离子聚合物、脂质、纳米颗粒、电穿孔、微注射、生物弹道、肽聚合物和细胞外囊泡。这些系统旨在有效地将 siRNA 运输到目标位置，并尽量减少不良反应。设计非病毒载体进行基因递送的兴趣从未减弱，因为相比病毒载体，它们在抗原特异性免疫反应方面的风险较低，尽管通常非病毒载体的转染效率较低。阳离子聚合物或脂质（例如 Lipofectamine® 2000）对裸 RNA 的包裹在商业产品中占据了重要地位，尽管这也带来了一定的细胞毒性和短期转染效果。
In recent advances, the design of nanomaterials for the safe and efficient delivery of siRNA is one of the challenging and rapidly growing areas of research since they have to overcome the commonly encountered biological barriers. Among those nanomaterials, carbon nanotubes (CNTs) which are rolled up seamless cylinders of graphene sheets have attracted great interests in the field of nanomedicine for the past decades as potential carriers for nucleic acid therapeutics such as siRNA. They have been proposed to easily cross the plasma membrane and to translocate directly into cytoplasm of target cells due to their needle-like structure. Although poor dispersity of CNTs in water is one of the limitations for most of their applications, they offer a structural advantage for further modification owning to their large surface area.
在近期研究中，纳米材料的设计用于安全高效地递送 siRNA 成为一个具有挑战性且快速发展的领域，这主要是因为它们需要克服常见的生物屏障。在众多纳米材料中，碳纳米管（CNTs）作为无缝卷曲的石墨烯圆柱体，近年来在纳米医学领域引起了广泛关注，成为核酸治疗（如 siRNA）的潜在载体。由于其针状结构，CNTs 被认为能够轻松穿越细胞膜，直接进入目标细胞的细胞质。尽管 CNTs 在水中的分散性较差是其应用的一大限制，但由于其大的表面积，CNTs 在进一步改性方面具有明显优势。
Particularly, covalent and non-covalent functionalization of CNTs have been designed for the purpose of siRNA delivery. The well-known transfection agent polyethyleneimine (PEI) was covalently functionalized onto CNTs and the resulting nanohybrids have shown superior siRNA silencing effect compared to the reference lipidic carrier in the presence of serum proteins. SiRNA complexed with ammonium functionalized CNTs via 1,3-dipolar cycloaddition of azomethine ylides were found to significantly suppress the growth of human lung tumor with intratumoral administration to the animal model, which makes carbon nanotubes potentially favorable for delivery siRNA therapeutics in vivo in comparison with liposome. In terms of more precise tailoring of CNTs for siRNA delivery, Prato et al. reported a direct correlation between amounts of cationic charges on CNTs which was covalently functionalized with quaternary amine dendrons and siRNA that accumulated into cell. The dendron-MWNT constructs have also been shown to complex siRNA and mediate its efficient intracellular delivery and biological activity with minimal induced cytotoxicity by controlling the number of polycationic functional groups on CNTs. Similarly, CNTs covalently functionalized with lipids and natural amino acid-based dendrimers also displayed effective protection for siRNA clearance and silencing for in vivo study. Others have also reported to use lipid for the functionalization of carbon nanotubes for siRNA delivery, but with noncovalent method. For instance, CNTs were noncovalently associated with a lipopolymer-PEI conjugation, in which the lipopolymer binds to CNTs via hydrophobic interaction, resulting in a better dispersity of CNTs in physiological conditions and the exposed PEI was used to offer binding site for siRNA. Unlike the commonly used strategy for complexing siRNA, which was through electrostatic interaction, siRNAs was also reported to couple with phospholipids functionalized CNTs via cleavable disulfide linkage, which can be cleaved by thiol reducing enzymes and promote the release of siRNA from lysosomal lipid vesicles to reach the cell cytosol.
特别地，CNTs 的共价和非共价功能化都是为了实现 siRNA 的递送。已知的转染试剂聚乙烯亚胺（PEI）被共价连接到 CNTs 上，所得到的纳米杂化物在含有血清蛋白的条件下显示出优于参考脂质载体的 siRNA 沉默效果。与氨基功能化的 CNTs 通过 1,3-偶极环加成反应结合的 siRNA，能显著抑制人肺肿瘤的生长，这使得碳纳米管在体内递送 siRNA 治疗方面优于脂质体。关于 CNTs 在 siRNA 递送中的精细调控，Prato 等人发现，季铵盐树状聚合物的阳离子电荷数量与细胞内积累的 siRNA 之间存在直接相关性。树状聚合物-多壁纳米管构建体已被证明能够有效地复合 siRNA，并以较低的细胞毒性介导其细胞内递送和生物活性，同时通过调控 CNTs 上的阳离子功能组数量来实现。类似地，共价功能化的 CNTs 与脂质和天然氨基酸基树状聚合物也能有效保护 siRNA，适用于体内研究。其他研究者还报告了使用脂质对碳纳米管进行非共价功能化，以递送 siRNA。例如，CNTs 与脂质聚合物-PEI 的结合通过疏水相互作用进行，从而在生理条件下改善了 CNTs 的分散性，并且暴露的 PEI 提供了 siRNA 的结合位点。与常用的通过静电作用复合 siRNA 的方法不同，siRNA 也被报告通过可裂解的二硫键与功能化的磷脂质 CNTs 结合，这种二硫键能够被硫醇还原酶切割，从而促进 siRNA 从溶酶体脂质囊泡释放到细胞质中。
To date, many methods have been explored to quantify the silencing efficiency of CNTs based siRNA delivery systems in vitro. Some fluorescently labeled siRNAs have been used to obtain quick estimates of delivery efficiency and localization. However, this approach is neither quantitative nor indicative of siRNA integrity and there is a risk of fluorescence quenching. Recently, a simple FRET electrophoresis method was developed by Tuttolomondo and Ditzel to dynamically evaluate serum siRNA stability and its interaction with the serum components, which is vital for in vivo administrations of siRNAs. Common techniques for studying transcriptional efficiency include Northern blotting, real-time PCR, microarray analysis, etc. Other methods to quantify the expression of the corresponding proteins are typically measured with western blot and flow cytometry. There is a need to develop reliable and easy-to-use assays to evaluate the binding and delivery efficiency of carbon nanotube-based systems.
到目前为止，已经探索了多种方法来定量 CNTs 基于 siRNA 递送系统的沉默效率。在体外实验中，使用了荧光标记的 siRNA 来快速估计递送效率和定位。然而，这种方法并不定量，也不能指示 siRNA 的完整性，且存在荧光淬灭的风险。最近，Tuttolomondo 和 Ditzel 开发了一种简单的 FRET 电泳方法，用于动态评估血清 siRNA 的稳定性及其与血清成分的相互作用，这对于 siRNA 在体内给药至关重要。研究转录效率的常用技术包括 Northern blotting、实时 PCR、微阵列分析等。其他定量相应蛋白表达的方法通常采用西方印迹和流式细胞术。需要开发可靠且易于使用的检测方法，以评估基于碳纳米管的系统的结合和递送效率。
This chapter describes the detailed protocols for siRNA binding and delivery quantification using gel electrophoresis and flow cytometry. To quantify siRNA binding, we have used cationic polymer-coated CNTs mixed with siRNA at different N/P ratios, quantified by gel electrophoresis. The siRNA binding of the corresponding free cationic polymer has also been studied where we observed better binding efficiency with cationic polymer functionalized CNTs than the free polymer. The optimal N/P ratio was then used to investigate the siRNA silencing in a model cell line. Commercialized transfection reagent Lipofectamine® 2000 was used as positive control. For quantification of the transfection efficiency, flow cytometry was used with a detailed protocol described in this chapter.
本章详细描述了使用凝胶电泳和流式细胞术定量 siRNA 结合和递送的协议。为定量 siRNA 结合，我们将阳离子聚合物涂层的 CNTs 与 siRNA 以不同的 N/P 比混合，并通过凝胶电泳进行定量。我们还研究了对应自由阳离子聚合物的 siRNA 结合情况，观察到与自由聚合物相比，阳离子聚合物功能化的 CNTs 具有更好的结合效率。随后使用最佳的 N/P 比来研究模型细胞系中的 siRNA 沉默。商业化转染试剂 Lipofectamine® 2000 作为阳性对照。在本章中详细描述了使用流式细胞术定量转染效率的协议。
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2.1 Preparation of siRNA Complexes siRNA 复合物的制备
1× phosphate-buffered saline (PBS): transfer 100 ml 10× PBS stock solution to 900 ml deionized H2O and autoclave at 120 °C, 20 min before use.1×磷酸盐缓冲盐水（PBS）：将 100 ml 的 10× PBS 储备液转移至 900 ml 去离子水中，在使用前在 120°C 下高压灭菌 20 分钟。
Bath sonicator (Ultrasonic cleaner, VWR).水浴超声清洗机（Ultrasonic cleaner, VWR）
RNaseZap™ (Invitrogen) to clean the workbench for gel electrophoresis assay (see Note 1).RNaseZap™（Invitrogen）用于清洁电泳实验的工作台（参见 注 1）
Cationic polymer (see Note 2).阳离子聚合物（参见 注 2）
Cationic polymer-CNT (see Note 3).阳离子聚合物-CNT（参见 注 3）
0.5 and 1.5 ml RNase-free microcentrifuge tubes, natural.0.5 ml 和 1.5 ml 无 RNase 微量离心管，中性材质
2.2 Gel Electrophoresis for siRNA Binding Capacity siRNA 结合能力的凝胶电泳
Agarose .琼脂糖
Sodium borate acid (SBA) buffer: mix 0.4 g of sodium hydroxide and 2.25 g of boric acid in 1 l deionized H2O and adjust the pH to 8.硼酸钠缓冲液（SBA）：将 0.4 g 氢氧化钠和 2.25 g 硼酸混合在 1 L 去离子水中，并调整 pH 至 8
Gel electrophoresis apparatus.凝胶电泳设备。
Gel loading dye and GelRed.凝胶加载染料和 GelRed
Bio-Rad ChemiDoc MP™ Imaging System.Bio-Rad ChemiDoc MP™成像系统
2.3 Cell Culture and Transfection 细胞培养与转染
RPMI-1640 medium , FBS (Fetal Bovine Serum), P/S (Penicillin-Streptomycin, 5000 U/ml), l-glutamine (GlutaMAX™), trypsin-EDTA (Thermo Scientific™).RPMI-1640 培养基，胎牛血清（FBS），青霉素-链霉素（P/S，5000 U/ml），l-谷氨酰胺（GlutaMAX™），胰蛋白酶-EDTA（Thermo Scientific™）
Full culture medium: RPMI-1640 supplied with 10% FBS, 1% P/S, and 1% l-glutamine (see Note 4).完全培养基：RPMI-1640 培养基中添加 10% FBS、1% P/S 和 1% l-谷氨酰胺（参见 注 4）
OPTI-MEM medium and Lipofectamine® 2000 (Thermo Scientific™).OPTI-MEM 培养基和 Lipofectamine® 2000（Thermo Scientific™）
T75 cell culture flask with filter.T75 细胞培养瓶（带过滤器）
Hemocytometer.血细胞计数板
Cell culture incubator.细胞培养培养箱
12-Well Clear TC-treated microplates .12 孔清晰 TC 处理微孔板
2.4 Antibody Staining and Flow Cytometry
Antibody for target and the corresponding isotype (Abcam®).目标抗体及其对应的同型对照（Abcam®）
Sodium azide (Sigma Aldrich).氟化钠（Sigma Aldrich）
Polystyrene round bottom 12 × 75 mm2 falcon tubes.聚苯乙烯圆底 12 × 75 mm² Falcon 管
BD FACSCalibur.BD FACSCalibur
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3.1 Preparation of siRNA Complexes siRNA 复合物的制备
Weigh 5 mg cationic polymer in a 7.5 ml glass vial.称取 5 mg 阳离子聚合物于 7.5 ml 玻璃瓶中。
Dissolve the cationic polymer with 5 ml of 1× PBS to make the stock concentration of 1 mg/ml.用 5 ml 的 1× PBS 溶解阳离子聚合物，制备浓度为 1 mg/ml 的储备液。
Apply sonication during the dissolution process and make sure the polymer is completely dissolved (see Note 5).在溶解过程中进行超声处理，确保聚合物完全溶解（见 注释 5）
Keep the cap of the vial sealed and stock the polymer solution in the fridge until use.封闭瓶盖，将聚合物溶液存放于冰箱中直至使用
Disperse cationic polymer-CNT in 1× PBS to make the final cationic polymer concentration of 1 mg/ml.将阳离子聚合物-CNT 分散于 1× PBS 中，以制备最终浓度为 1 mg/ml 的阳离子聚合物
Sonicate the sample until it completely disperses in PBS and keep the cationic polymer-CNT dispersion in the fridge until use (see Note 6).超声处理样品，直到其完全分散于 PBS 中，并将阳离子聚合物-CNT 分散液存放于冰箱中直至使用（见 注释 6）
Calculation of N/P ratio for siRNA complex. The N/P ratio is the ratio of amine (N, nitrogen) groups on positively-charged polymer to the phosphate (P) groups on negatively-charged nucleic acid. It is important to determine the N/P of a cationic polymer-based nucleic acid complex as this character can influence many other properties such as the surface charge, size, and stability of the nucleic acid complex. The calculation of N/P ratio is described as follows and the units used are based on the international system of units. Phosphate moles of siRNA: depending on the sequence, siRNA can have 40–42 phosphate atoms per molecule in a 20–21 bp. The total phosphate atoms can be calculated as (n × P, where n is the total number of moles of siRNA taken and P is the phosphate atoms per molecule). Nitrogen moles of cationic polymer: the total moles of nitrogen atoms can be calculated as (M × V × N, where M is the molarity of cationic polymer stock solution based on the repeat unit, V is the volume of cationic polymer taken and N is the number of nitrogens in one monomer unit of cationic polymer.) Thus, the N/P ratio can be calculated as: (M × V × N)/(n × P). Usually, for siRNA loading and transfection assays, the M, N, n, and P are known, and thus the V can be calculated based on the desired N/P.siRNA 复合物的 N/P 比计算。N/P 比是指阳离子聚合物上阳离子（氮，N）基团与负电荷核酸上的磷（P）基团的比值。确定基于阳离子聚合物的核酸复合物的 N/P 比非常重要，因为这一特性会影响复合物的表面电荷、尺寸和稳定性等其他多个特性。N/P 比的计算如下，其单位基于国际单位制。siRNA 的磷酸盐摩尔数：根据序列，siRNA 每个分子可以有 40-42 个磷酸盐原子（每个 20-21 bp）。总磷酸盐原子数可计算为 (n × P，其中 n 是所取 siRNA 的总摩尔数，P 是每个分子的磷酸盐原子数）。阳离子聚合物的氮摩尔数：氮原子的总摩尔数可计算为 (M × V × N，其中 M 是基于重复单元的阳离子聚合物储备液的浓度，V 是所取阳离子聚合物的体积，N 是阳离子聚合物单体中氮的数量）。因此，N/P 比可计算为：(M × V × N)/(n × P)。通常，在 siRNA 装载和转染实验中，M、N、n 和 P 是已知的，从而可以根据所需的 N/P 计算V
After calculating the N/P ratio, the siRNA solution (in 1× PBS) and polymer solution/polymer-CNT suspension (in 1× PBS) are prepared separately in two Eppendorf tubes.计算 N/P 比后，将 siRNA 溶液（在 1× PBS 中）和聚合物溶液/聚合物-CNT 悬浮液（在 1× PBS 中）分别准备在两个 Eppendorf 管中
After 5 min incubation at R.T., the cationic polymer solution/cationic polymer-CNT suspension is added to the siRNA solution dropwise and mixed gently by tapping the tube.在室温下孵育 5 分钟后，将阳离子聚合物溶液/阳离子聚合物-CNT 悬浮液逐滴加入 siRNA 溶液中，并轻轻摇动管子混匀
The siRNA complex is left at R.T. for another 20 min incubation before performing gel electrophoresis and transfection study.将 siRNA 复合物在室温下再孵育 20 分钟，然后进行凝胶电泳和转染研究
3.2 Gel Electrophoresis for siRNA Binding Capacity siRNA 结合能力的凝胶电泳
Add 2 g agarose powder to 100 ml 1× SBA in a conical flask (see Note 7).将 2 g 琼脂糖粉末加入 100 ml 的 1× SBA 中，放入锥形瓶中（见 注释 7）
Microwave agarose mixture at full power for 2 min total. Microwave continuously for the first 1 min (see Note 8), stop, and swirl flask.在微波炉中以全功率加热琼脂糖混合物，总共加热 2 分钟。前 1 分钟连续微波（见 注释 8），然后停止并摇晃锥形瓶。
Every subsequent 15 s, swirl flask to dissolve agarose clumps until agarose completely dissolves (see Note 9).之后每 15 秒摇晃一次锥形瓶，直至琼脂糖完全溶解（见 注释 9）。
Pour the mixture into gel cast, add comb, and leave to set for 30 min (R.T.) or 10 min (4 °C).将混合物倒入凝胶铸模中，添加梳子，室温下静置 30 分钟（或在 4 °C 下静置 10 分钟）以使其凝固。
Transfer agarose and gel cast into running tank.将琼脂糖和凝胶铸模转移到电泳槽中。
Fill tank with 1× SBA buffer until just covering gel (1–2 mm above the gel) (see Note 10).用 1× SBA 缓冲液填充槽，直到刚好覆盖凝胶（高出凝胶 1-2 mm）（见 注释 10）
Add 1μl of 6× Gel Loading Dye + GelRed to 5μl of sample (siRNA complex or free siRNA in step 4, Subheading 3.1 above). Load 6μl sample/ladder into wells on agarose gel.向 5μl 样品（siRNA 复合物或步骤 4 中的游离 siRNA）中添加 1μl 的 6×凝胶加载染料+GelRed。将 6μl 样品/梯度装入琼脂糖凝胶的孔中。
Run gel at constant 225 V for 12 min (see Note 11).在 225 V 恒定电压下运行凝胶 12 分钟（见 注释 11）。
Image the gel with Bio-Rad ChemiDoc MP™ Imaging System.使用 Bio-Rad ChemiDoc MP™成像系统对凝胶进行成像。
Calculation of siRNA binding percentage.计算 siRNA 结合百分比。
The gel image (Fig. 1a) is analyzed by Image Lab™ software (see Note 12). The siRNA binding percentage (Fig. 1b) is calculated as follows:凝胶图像（图 1a）通过 Image Lab™软件进行分析（见 注释 12）。siRNA 结合百分比（图 1b）计算公式如下：
s i R N A b i n d i n g % = 1 0 0 − i n t e n s i t y s i R N A c o m p l e x i n t e r n s i t y f r e e s i R N A × 1 0 0
Fig. 1 Quantifications of siRNA binding capacity . (a) Representative images of gel electrophoresis. (b) Analysis of band intensity for siRNA binding with cationic polymer and cationic polymer coated CNT at different N/P ratios compared with free siRNA sample. Values are expressed as mean ± SD, where n = 3, statistical analysis was done on cationic polymer and cationic polymer-CNT (p** < 0.01, p*** < 0.001)
3.3 Cell Culture 细胞培养
The cells used for transfection assay are dependent on specific experimental design. The following protocol describes the culture of B16-F10 cells (murine melanoma cell line), which is used later in the transfection study.
用于转染实验的细胞取决于具体实验设计。以下协议描述了 B16-F10 细胞（小鼠黑色素瘤细胞系）的培养，该细胞系将在后续转染研究中使用。
B16-F10 cells were cultured in full-culture medium. Upon confluency, discard the old medium, rinse the cells with 10 ml PBS once, and add 1 ml trypsin-EDTA to cells for 3 min in an incubator (see Note 13).在完全培养基中培养 B16-F10 细胞。待细胞生长至汇合状态，弃去旧培养基，使用 10 ml PBS 冲洗细胞一次，并加入 1 ml 胰蛋白酶-EDTA 于细胞中在培养箱中孵育 3 分钟（见 注释 13）
Add 10 ml of fresh medium to deactivate trypsinization.添加 10 ml 新鲜培养基以使胰蛋白酶失活
Collect cells in a 20 ml universal tube and centrifuge at 400 × g for 5 min.将细胞收集到 20 ml 通用管中，离心 400 × g 5 分钟
Resuspend the cell pellet in 10 ml complete medium and count the cell number with hemocytometer.用 10 ml 完全培养基重悬细胞沉淀，并用血细胞计数板计数
Adjust cell suspension to 150,000 cells/ml in complete medium.将细胞悬液调整至 150,000 细胞/ml 的完全培养基
Seed 75,000 cells per well in 12-well plates (around 60% confluency) and incubate for 24 h in an incubator before transfection assay (see Note 14).每孔接种 75,000 细胞于 12 孔板中（约 60%汇合），并在转染实验前在培养箱中孵育 24 小时（见 注释 14）
3.4 Cell Transfection 细胞转染
siRNA (30 nM) is used for transfection assay . Serum-free OPTI-MEM medium is used for preparing siRNA complex and transfection (see Note 15).
siRNA（30 nM）用于转染实验。使用无血清 OPTI-MEM 培养基制备 siRNA 复合物和转染（见 注释 15）。
Prepare 100μl of siRNA OPTI-MEM solution in a 1.5 ml Eppendorf tube (see Note 16).在 1.5 ml Eppendorf 管中准备 100μl 的 siRNA OPTI-MEM 溶液（见 注释 16）。
Prepare 100μl of cationic polymer/cationic polymer-CNT solution in OPTI-MEM according to the desired N/P ratio (10 in this case).按照所需的 N/P 比（本例中为 10），在 OPTI-MEM 中准备 100μl 阳离子聚合物/阳离子聚合物-CNT 溶液。
Prepare Lipofectamine® 2000 solution in OPTI-MEM medium according to the manufacturer’s instruction as positive control.根据制造商的说明，在 OPTI-MEM 中准备 Lipofectamine® 2000 溶液作为阳性对照。
After 5 min incubation at R.T., the cationic polymer solution/cationic polymer-CNT suspension/Lipofectamine solution are added to the siRNA solution dropwise and mixed gently by tapping the tube.在室温下孵育 5 分钟后，阳离子聚合物溶液/阳离子聚合物-CNT 悬浮液/Lipofectamine 溶液逐滴加入 siRNA 溶液中，并轻轻摇动管子混匀。
The siRNA complex (200μl) is left incubation at R.T. for another 20 min.siRNA 复合物（200μl）在室温下孵育 20 分钟。
The old medium in 12-well plate containing B16-F10 cells are removed and 1 ml of PBS is added to rinse the cells.移除含有 B16-F10 细胞的 12 孔板中的旧培养基，并添加 1 ml PBS 以冲洗细胞。
800μl of serum-free OPTI-MEM medium is added to the well.向孔中加入 800μl 无血清 OPTI-MEM 培养基。
200μl of siRNA complex is added dropwise to the well.逐滴添加 200μl siRNA 复合物于孔中。
For blank cells, 1 ml serum-free OPTI-MEM medium is added 对于空白细胞，添加 1 ml 无血清 OPTI-MEM 培养基。
Incubate the cell for 4–6 h at a cell culture incubator.在细胞培养箱中孵育 4-6 小时。
The serum-free OPTI-MEM medium is changed with full culture medium for a further 24–48 h culture before harvesting. 将无血清 OPTI-MEM 培养基更换为完全培养基，并继续培养 24-72 小时以进行转染效果评估。
After 24–48 h culture, the old medium is removed, and the cells are rinsed with 1 ml PBS.在 24–48 小时培养后，移除旧培养基，用 1 ml PBS 冲洗细胞。
100μl of trypsin-EDTA is used to trypsinize the cell for 3 min in a cell culture incubator.使用 100μl 胰蛋白酶-EDTA 在细胞培养箱中对细胞进行胰蛋白酶处理 3 分钟。
After 3 min, 1 ml of complete medium is added to each well, and cells are collected in polystyrene round bottom 12 × 75 mm2 falcon tubes for further analysis.处理 3 分钟后，每孔添加 1 ml 完全培养基，并将细胞收集于聚苯乙烯圆底 12 × 75 mm²的法尔康管中，以备进一步分析。
3.5 Antibody Staining for Flow Cytometry Analysis 流式细胞术分析的抗体染色
The antibody staining protocol (e.g., antibody concentration, staining time, temperature) should be optimized according to the certain types of cells and antibody.
抗体染色方案（例如，抗体浓度、染色时间、温度）应根据特定类型的细胞和抗体进行优化。
The cells harvested in Subheading 3.4 are washed with PBS and resuspended in 100μl ice-cold PBS with 3% FBS.将在 3.4 小节中收集的细胞用 PBS 洗涤，并重悬于 100μl 冰冷 PBS 中，加入 3% FBS。
Transfer 50μl of cell suspension to a new tube for isotype staining.将 50μl 细胞悬液转移至新管中用于同型对照染色。
Add 0.1μg/ml of conjugated primary antibody (or the corresponding isotype) to the cell suspension.向细胞悬液中添加 0.1μg/ml 的标记主抗体（或相应的同型对照）。
Incubate at R.T. for 20 min in dark.在室温下避光孵育 20 分钟。
Wash the cells three times with PBS and centrifuge at 400 × g for 5 min (see Note 17).用 PBS 洗涤细胞三次，并以 400 × g离心 5 分钟（见 注释 17）。
Resuspend the cells in 200μl of ice-cold PBS with 10% FBS, 1% sodium azide.将细胞重悬于 200μl 冰冷 PBS 中，加入 10% FBS 和 1%叠氮化钠。
Keep the cells in fridge until analysis.将细胞放置在冰箱中直至分析。
For best result, analyze the cells with flow cytometry as soon as possible.为了获得最佳结果，尽快用流式细胞仪分析细胞。
Fixation of the cells may be needed for cells kept longer than 1 h before analysis (see Note 18).对于在分析前存放超过 1 小时的细胞，可能需要进行固定处理（见 注释 18）。
3.6 Flow Cytometry Analysis for Target Protein Expression 目标蛋白表达的流式细胞术分析
Samples collected from Subheading 3.5 are analyzed with flow cytometry (BD FACSCalibur, using CellQuest software) using 488 nm excitation and a 585 nm bandpass filter for fluorescein detection (see Note 19).从 3.5 小节中收集的样品使用流式细胞仪（BD FACSCalibur，使用 CellQuest 软件）进行分析，采用 488 nm 激发波长和 585 nm 带通滤光片进行荧光素检测（见 注释 19）。
10,000 events per sample are analyzed.每个样品分析 10,000 个事件。
Isotype controls are also analyzed for each sample.每个样品也分析同型对照。
3.7 Data Analysis 数据分析
Subsequent analysis of the target expression is done using FlowJo software (TreeStar, Ashland, OR).
目标表达的后续分析使用 FlowJo 软件（TreeStar，Ashland，OR）进行。
A subset of data can be defined through a gate, which is a numerical or graphical boundary that can be used to define the characteristics of particles to include for further analysis. A gate is set on the FSC vs. SSC plot to exclude any debris and allow further analysis of the viable cell population.可以通过设置门（gate）来定义数据的子集，门是用于定义颗粒特征的数值或图形边界，以便进行进一步分析。在 FSC 与 SSC 图上设置门以排除任何碎片，并允许进一步分析活细胞群体。
Using the viable cells gate, another gate of target expression is set on the isotype stained sample to ensure the observed staining is due to specific antibody binding to the target and is not an artifact.使用活细胞门，基于同型对照样本设置目标表达的另一个门，以确保观察到的染色是由于特异性抗体与目标结合，而非伪影。
The same gate from the isotype sample is applied to the corresponding sample to determine the expression of the target, the representative dot plots are shown in Fig. 2.将同型样本的门应用于相应样本，以确定目标的表达，代表性的点图如图 2 所示。
The mean fluorescence intensity (MFI) is exported and relative target expression % (Fig. 3) is calculated as follows:导出平均荧光强度（MFI），并计算相对目标表达百分比（图 3），计算公式如下：
R e l a t i v e t a r g e t e x p r e s s i o n % = M F I s a m p l e M F I b l a n k c e l l s × 1 0 0
Fig. 2 Representative flow cytometry dot plots for B16-F10 cell transfection with different siRNA complexes, the gate for the target expression was corrected with the corresponding isotype for each sample
Fig. 3 B16-F10 cell transfection with different siRNA complexes quantified by flow cytometry with both mean fluorescent intensity (MFI) and relative target expression % compared with blank cells with siRNA concentration of 30 nM. Values are expressed as mean ± SD, where n = 3. Statistical analysis was done against blank cells (p* < 0.01, p**** < 0.001)
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Spray the work bench with RNaseZap each time to minimize the degradation of the nucleic acids.每次在工作台上喷洒 RNaseZap，以最小化核酸的降解。
Cationic polymer is the same polymer used to functionalize carbon nanotube samples and it may vary according to different purposes. Here we use free cationic polymer as a control to cationic polymer-CNT samples.阳离子聚合物是用于功能化碳纳米管样品的相同聚合物，可能根据不同用途有所变化。在此，我们使用自由阳离子聚合物作为对照，与阳离子聚合物-CNT 样品进行比较。
The properties of cationic polymer used to functionalize CNT and the method of functionalization may vary according to different purposes.用于功能化 CNT 的阳离子聚合物的性质和功能化方法可能根据不同用途有所不同。
Cell culture medium is prepared with 500 ml medium and added supplements for the whole bottle. Aliquots are taken each time for medium warm up instead of warming the whole bottle.细胞培养基使用 500 ml 培养基和添加补充剂准备整瓶。每次取出一定量进行培养基加热，而不是加热整瓶。
Sonicate the cationic polymer and ensure it is fully dissolved in PBS.超声波处理阳离子聚合物，确保其在 PBS 中完全溶解。
Sonicate the cationic polymer-CNT samples every time for at least 10 min before use to ensure the well dispersity of the sample.每次使用前，阳离子聚合物-CNT 样品需超声处理至少 10 分钟，以确保样品良好的分散性。
Agarose powder must be uniformly dispersed in buffer prior to hydration to avoid clumping. To determine gel volume using the following estimation: Gel volume = surface area of the casting chamber × gel depth (3–5 mm thick).在水合前，琼脂糖粉末必须在缓冲液中均匀分散，以避免结块。使用以下估算来确定凝胶体积：凝胶体积 = 铸模腔的表面积 × 凝胶深度（厚度为 3–5 mm）。
For smaller or larger volumes, increase or decrease heating times proportionally to volume size.对于较小或较大的体积，相应地增加或减少加热时间。
Heating times will vary depending on the wattage of your microwave oven, size of the flask used, and the agarose concentration. Caution: Handle the hot flask very carefully. Microwaved solutions may become superheated and boil over when moved or touched.加热时间会因微波炉的功率、所用烧瓶的大小以及琼脂糖浓度而有所不同。注意：在处理热烧瓶时要非常小心，微波加热的溶液可能会过热，当移动或触碰时会沸腾溢出。
Allow the agarose solution to cool to ~50–55 °C before pouring the gel into the prepared casting stand. This will result in a gel with a more uniform pore size and prevent warping of the gel apparatus.在将琼脂糖溶液倒入准备好的铸模支架之前，允许其冷却至约 50–55 °C。这将使凝胶具有更均匀的孔径，并防止凝胶设备的变形。
The voltage and time for gel running may vary according to sample properties and gel apparatus.凝胶运行的电压和时间可能根据样品性质和凝胶设备而有所不同。
Use the converted gel image (white background and gray bands) for siRNA binding analysis.使用转换后的凝胶图像（白色背景和灰色带）进行 siRNA 结合分析。
Trypsinization time may vary for different cell types.胰蛋白酶处理时间可能因不同细胞类型而有所不同。
The seeded cell density may vary with different cell type and experiment design. For low-passage cells, make sure cells are healthy and around 80–90% viable before transfection. For high-passage cells, adjust the seeded cell number accordingly and take into account also with the cytotoxicity of the transfection agents. The assay timepoint should also be considered—lower cell densities for long-term assays and higher cell numbers for short-term experiments.种子细胞密度可能因不同细胞类型和实验设计而异。对于低传代细胞，确保细胞健康且存活率在 80–90%之间；对于高传代细胞，适当调整种子细胞数量，同时考虑转染试剂的细胞毒性。实验的时间点也应考虑——长期实验需较低的细胞密度，而短期实验需较高的细胞数量。
siRNA concentration and transfection medium used may vary according to the design of the experiment and also the cell type. Do not add antibiotics to the medium during the transfection as this reduces the transfection efficiency and causes cell death.siRNA 浓度和使用的转染培养基可能根据实验设计和细胞类型有所不同。在转染过程中，请勿在培养基中添加抗生素，因为这会降低转染效率并导致细胞死亡。
100μl siRNA solution is prepared for transfection done in 12-well plate. The amount of siRNA solution may vary according to different well plate applied, and the amount of cationic polymer should also be changed accordingly.为 12 孔板转染准备 100μl siRNA 溶液。根据所应用的不同孔板，siRNA 溶液的量可能有所不同，阳离子聚合物的量也应相应调整。
This step is to remove any unbound free antibody extensively.此步骤是为彻底去除任何未结合的自由抗体。
For extended storage as well as for greater flexibility in planning time on the cytometer, resuspend cells in 1–4% paraformaldehyde to prevent deterioration.为了延长储存时间以及在流式细胞仪上规划时间的灵活性，使用 1–4%多聚甲醛重悬细胞，以防止细胞变质。
The excitation wavelength and filter may vary according to the specific dye and flow cytometer used.激发波长和滤光片可能根据特定染料和所用流式细胞仪而有所不同。
