Hiroyuki Koide
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Small interfering RNA (siRNA) is a novel therapeutic modality for the treatment of intractable diseases; however, the development of a useful siRNA delivery vector is imperative for clinical use. Since siRNA works in the cytoplasm, the ability of the carrier to escape destruction in the endosomes is a highly required characteristic for the induction of a high knockdown effect. Here, we describe the step-by-step procedure for the evaluation of high endosomal escapability. The vector that has pH-responsive characteristics at around pH = 6.2–6.5 is important for the high endosomal escape.
小干扰 RNA（siRNA）是一种新兴的治疗方式，主要用于治疗难治性疾病，但有效的 siRNA 递送载体的开发对其临床应用至关重要。由于 siRNA 在细胞质中发挥作用，载体必须具备在内体中逃避降解的能力，这一特性对于实现高效的基因沉默至关重要。本文介绍了评估高内体逃逸能力的详细步骤。具有在 pH 值约为 6.2 至 6.5 范围内响应的载体，对于提高内体逃逸效果具有重要意义。
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Small interfering RNA (siRNA) specifically binds to its target mRNA in the cytoplasm after making a complex with the RNA-induced silencing complex (RISC), which cleaves the target mRNA in a sequence-dependent manner. Because of its high specificity, siRNA is expected to be an attractive medicine for the treatment of intractable diseases such as cancer and inflammation. However, since siRNA is easily degraded by RNase and hard to penetrate the cellular membrane because of its high molecular weight and negative charge, the delivery of siRNA into the cytoplasm without a vehicle is extremely difficult. Therefore, the development of a useful vector that allows high cellular uptake, endosomal escape, and siRNA release in the cytoplasm is required for proper siRNA delivery. Recently, it was reported that only a few percent of siRNA molecules escape from the endosomes by use of conventional cationic lipids. Thus, an increase in endosomal escapability is a critical necessity for siRNA delivery and induction of a high knockdown effect.
小干扰 RNA（siRNA）在与 RNA 诱导沉默复合体（RISC）结合后，能够特异性地识别并切割靶 mRNA，其过程具有序列依赖性。由于 siRNA 的高特异性，它被认为是一种有潜力的治疗药物，适用于癌症和炎症等难治性疾病。然而，siRNA 容易受到核糖核酸酶（RNase）的降解，并且由于其高分子量和负电荷，难以穿透细胞膜，这使得在没有载体的情况下将 siRNA 递送至细胞质变得极为困难。因此，开发一种有效的载体，以实现高效的细胞摄取、内体逃逸和细胞质中 siRNA 的释放，成为了确保 siRNA 成功递送的必要条件。最近的研究表明，传统阳离子脂质只能让少量 siRNA 分子逃逸出内体。因此，提高内体逃逸能力对于 siRNA 的递送和实现有效的基因沉默至关重要。
Here, we show how to design pH-responsive polycationic liposomes to overcome the endosomal escapability problem. We prepared polycation liposomes containing a dioleylphosphate-diethylenetriamine conjugate (DOP-DETA, Fig. 1) , a pH-responsive polycationic lipid, were prepared and complexed with siRNA (lipoplex). We prepared two lipoplexes , a high liposome concentration lipoplex (cationic liposome:siRNA = 7000:1 as a molar ratio, High-lipoplex) and a low liposome concentration lipoplex (polycationic liposome:siRNA = 700:1 as a molar ratio, Low-lipoplex). The titration curve of each lipoplex was determined by 2-(p-toluidino)-6-napthalene sulfonic acid (TNS) assay (Fig. 2). The fluorescence intensity of TNS molecules reflects the ionization of delivery vehicles for siRNA. The TNS assay is an easy method to evaluate pH responsiveness of siRNA vehicles and obtain pKa values. To demonstrate whether pH-responsive lipoplex shows endosomal escapability, the localization of polycation liposomes, siRNA, and endosomes in cells was observed by confocal laser scan microscopy (Fig. 3). By using this method, we can clearly observe whether siRNA escape from the endosome or not, which allows the evaluation of endosomal escapability of siRNA vehicles.
在本研究中，我们展示了如何设计 pH 响应性的聚阳离子脂质体，以解决内体逃逸的问题。我们制备了含有二油酰磷酸-二乙烯三胺偶联物（DOP-DETA，图 1）的聚阳离子脂质体，这种脂质在 pH 约 6.2 至 6.5 时具有响应性，并与 siRNA 形成复合物（脂质复合物）。我们制备了两种不同浓度的脂质复合物：高浓度脂质复合物（阳离子脂质体：siRNA = 7000:1 的摩尔比）和低浓度脂质复合物（聚阳离子脂质体：siRNA = 700:1 的摩尔比）。使用 2-（p-甲苯基氨基）-6-萘磺酸（TNS）测定每种脂质复合物的滴定曲线（图 2）。TNS 分子的荧光强度能够反映出 siRNA 递送载体的电离情况。TNS 测定方法简单易行，可用于评估 siRNA 载体的 pH 响应性并获得 pKa 值。为了验证 pH 响应性脂质复合物的内体逃逸能力，我们使用共聚焦激光扫描显微镜观察了细胞内聚阳离子脂质体、siRNA 和内体的分布（图 3）。这种方法使我们能够清晰地判断 siRNA 是否成功逃离内体，从而评估 siRNA 载体的内体逃逸能力。
Fig. 1 Chemical structure of DOP-DETA
Fig. 2 The surface pKa value of polycationic liposome and lipoplex . TNS was mixed with polycationic liposome alone (without siRNA), Low-lipoplex or High-lipoplex in a series of buffers with pH ranging from 4.0 to 10.0. Changes in fluorescence values are shown. The apparent pKa value of each sample is the pH value at which TNS fluorescence is half of the maximum fluorescence. The calculated pKa values for polycationic liposome were 6.7 and 9.3
Fig. 3 Intracellular distribution of polycationic liposomes and siRNA. Cells were imaged at 9 h after the transfection. The distribution of FITC-siRNA and DiD-labeled polycationic liposomes taken up into the cells was observed by use of confocal laser-scanning microscopy . Green; siRNA, blue; lysosome, red; liposome. Scale bars indicate 50 μm
From these results and those reported by others, we conclude that pH-responsiveness of siRNA vehicles is a critical factor for high endosomal escapability. We anticipate that the present procedures will provide helpful information for the development of useful siRNA delivery vehicles.
综上所述，包括其他研究的结果在内，我们认为 siRNA 载体的 pH 响应性是实现高效内体逃逸的关键因素。我们相信，本研究所提出的规程将为开发有效的 siRNA 递送载体提供重要的参考信息。
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Prepare all solutions with RNase-free water and analytical grade reagents. Store all reagents at 4 °C.
在准备所有溶液时，请使用无 RNase 水和分析纯试剂，并将所有试剂储存于 4°C。
2.1 Preparation of Polycationic Liposome and siRNA Complex
10 mM Dipalmitoylphosphatidylcholine (DPPC) in chloroform. 10 mM 二棕榈酰磷脂酰胆碱（DPPC）溶于氯仿中
10 mM Cholesterol in chloroform.10 mM 胆固醇溶于氯仿中
10 mM Dioleylphosphate-diethylenetriamine conjugate (DOP-DETA). 10 mM 二油酰磷酸-二乙烯三胺结合物（DOP-DETA）
1 mM 1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt (DiD) Store at −20 °C (see Note 1). 1 mM 1,1′-二八烷基-3,3,3′,3′-四甲基吲哚二氟代卡宾盐（DiD），储存于−20°C（见 注释 1）
RNase-free water: Add 1 mL diethylpyrocarbonate (DEPC) to 1 L ultrapure water and stir overnight to inactivate any RNase. Then, remove the remaining DEPC by autoclaving.制备无 RNase 水：向 1 L 超纯水中加入 1 mL 的二乙基焦磷酸（DEPC），搅拌过夜以失活 RNase，然后通过高压灭菌去除剩余的 DEPC
siRNA stock solution: Add DEPC-treated water to siRNA to prepare a 100 μM siRNA solution. Store at −20 °C (see Note 2).siRNA 母液：使用 DEPC 处理的水稀释 siRNA，制备 100 μM 的 siRNA 溶液，储存于−20°C（见 注释 2）
Extrusion filter: polycarbonate membrane.挤出过滤器：聚碳酸酯膜。
Extruder.挤出器
2.2 2-(p-Toluidino)-6-napthalene sulfonic acid (TNS) Assay 2-(p-托氨基)-6-萘磺酸（TNS）测定
25 mM Citric acid monohydrate 150 mM sodium chloride buffer adjusted the pH to 4.0, 4.5, 5.0, 5.5, and 6.0 with NaOH.25 mM 柠檬酸单水合物 150 mM 氯化钠缓冲液，pH 用 NaOH 调整至 4.0、4.5、5.0、5.5 和 6.0
20 mM Disodium hydrogen phosphate anhydrous 150 mM sodium chloride buffer adjusted the pH to 6.5, 7.0, and 7.5 with HCl.20 mM 无水磷酸氢二钠 150 mM 氯化钠缓冲液，pH 用 HCl 调整至 6.5、7.0 和 7.5
20 mM Ammonium acetate 150 mM sodium chloride buffer adjusted the pH to 8.0, 8.5, 9.0, 9.5, and 10.0 with NaOH (see Note 3).20 mM 醋酸铵 150 mM 氯化钠缓冲液，pH 用 NaOH 调整至 8.0、8.5、9.0、9.5 和 10.0（见 注释 3）
0.6 mM TNS dissolved in DMSO .0.6 mM TNS 溶解于 DMSO 中
96-Well black plates.96 孔黑板
2.3 Confocal Laser Scan Microscopy 共聚焦激光扫描显微镜
Cell culture medium : D-MEM/Ham’s F-12 medium containing 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin G, and 100 μg/mL streptomycin. For lysosome and endosome staining, prepare the medium without FBS but containing penicillin and streptomycin (see Note 4).细胞培养基：D-MEM/Ham’s F-12 培养基，含 10%热灭活胎牛血清（FBS）、100 U/mL 青霉素 G 和 100 μg/mL 链霉素。用于溶酶体和内体染色时，准备不含 FBS 但含青霉素和链霉素的培养基（见 注释 4）
siRNA observation: Fluorescein isothiocyanate (FITC)-labeled siRNAs at the 3-prime end of the antisense strand.siRNA 观察：在反义链的 3'末端标记荧光素异硫氰酸酯（FITC）标记的 siRNA
Imaging plate: 24-well black plates.成像板：24 孔黑板
Lysosome staining: Add 1 μL of Lysotracker Red DND-99 to 1 mL of the medium containing without FBS but containing penicillin and streptomycin.溶酶体染色：向不含 FBS 但含有青霉素和链霉素的培养基中添加 1 μL 的 Lysotracker Red DND-99
Wash solution: PBS containing 30 U/mL heparin.洗涤溶液：含 30 U/mL 肝素的 PBS
Cell fix solution: 4% paraformaldehyde dissolved in PBS (see Note 5).细胞固定溶液：4%多聚甲醛溶解于 PBS 中（见 注释 5）
Block solution: 3% bovine serum albumin (BSA) and 0.1% saponin-containing PBS (see Note 6).阻断溶液：含 3%牛血清白蛋白（BSA）和 0.1%皂素的 PBS（见 注释 6）
Nuclear staining: Add 10 μL of 10 μg/mL 4′,6-diamidino-2-phenylindole (DAPI) to 1 mL of PBS containing 3% BSA and 0.1% saponin.核染色：向含 3% BSA 和 0.1%皂素的 PBS 中添加 10 μL 的 10 μg/mL 4′,6-二脒基-2-苯基吲哚（DAPI）
Mounting reagent: PERMAFLUOR .封装试剂：PERMAFLUOR
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3.1 Preparation of Polycationic Liposomes and siRNA Complexes 多阳离子脂质体和 siRNA 复合物的制备
Add 120 μL of 10 mM DOP-DETA , 120 μL of 10 mM DPPC, and 60 μL of 10 mM cholesterol in chloroform to a 50-mL flask using a microsyringe. For the preparation of fluorescence-labeled polycation liposomes, 30 μL of 1 mM DiD is added to the lipid mixture under shielding from light. Add 30 mL of tert-butyl alcohol to the flask and mix gently (see Note 7).使用微量注射器将 120 μL 的 10 mM DOP-DETA、120 μL 的 10 mM DPPC 和 60 μL 的 10 mM 胆固醇加入 50 mL 的烧瓶中。为了制备荧光标记的多阳离子脂质体，在脂质混合物中添加 30 μL 的 1 mM DiD，需在避光条件下进行。然后向烧瓶中加入 30 mL 的tert-丁醇，轻轻混合（见 注释 7）。
Evaporate the chloroform with a rotary evaporator (see Note 8）. Freeze the lipid solution by using liquid nitrogen. Dry the frozen lipid solution with a freeze dryer for complete drying.使用旋转蒸发器蒸发氯仿（见 注释 8）。利用液氮将脂质溶液冷冻，然后使用冻干机彻底干燥脂质。
Add 3 mL of RNase-free water to the dried lipid powder to prepare 3 mM (total lipid concentration) polycationic liposomes (see Note 9). Repeat cycles of heating at 60 °C and vortexing 3 times, each for a few minutes (see Note 10).向干燥的脂质粉末中加入 3 mL 无 RNase 水，制备 3 mM 的多阳离子脂质体（见 注释 9）。反复加热至 60 °C 并涡旋 3 次，每次几分钟（见 注释 10）。
With an extruder device, extrude the polycationic liposomes 10 times through a polycarbonate membrane filter having a pore size of 100 nm (see Note 11).使用挤出设备，将多阳离子脂质体通过孔径为 100 nm 的聚碳酸酯膜过滤 10 次（见 注释 11）。
Incubate sized polycationic liposomes and siRNA (liposome:siRNA molar ratio = 7000:1 or 700:1) for 30 min at 37 °C to prepare High-lipoplex and Low-lipoplex (see Note 12). For confocal imaging, use siRNA conjugated with a fluorescent molecule such as fluoresceinisothiocyanate (FITC) at the 3-prime end of the antisense strand.将制备好的多阳离子脂质体与 siRNA（脂质体摩尔比为 7000:1 或 700:1）在 37 °C 下孵育 30 分钟，以制备高脂质体复合物（High-lipoplex）和低脂质体复合物（Low-lipoplex）（*见 注释 12）。用于共聚焦成像时，使用在反义链的 3'末端与荧光分子如荧光素异硫氰酸酯（FITC）偶联的 siRNA。
Dilute the prepared liposomes to 0.15 mM with RNase-free water. Then, measure their particle size, polydispersity index (PDI), and ζ-potential (see Note 13).使用无 RNase 水将制备好的脂质体稀释至 0.15 mM。然后测量其颗粒大小、聚合度指数（PDI）和ζ-电位（见 注释 13）。
3.2 Measurement of pKa of Liposome 脂质体的 pKa 测定
Add 20 μL of 2 mM liposomes to 1.96 mL of pH-adjusted buffer (pH 4.0–10.0) and incubate the liposomes for 20 min at room temperature (final lipid concentration: 20 μM). 将 20 μL 的 2 mM 脂质体加入 1.96 mL 经过 pH 调整的缓冲液（pH 4.0–10.0），在室温下孵育脂质体 20 分钟（最终脂质浓度：20 μM）。
After the incubation, add 20 μL of 0.6 mM TNS dissolved in DMSO to the mixture (final TNS concentration: 6 μM). Then, mix by vortexing.孵育后，向混合物中加入 20 μL 的 0.6 mM TNS（溶于 DMSO）（最终 TNS 浓度：6 μM），并进行涡旋混合。
Add 200 μL of the mixture to a 96-well black plate.将 200 μL 的混合物转移至 96 孔黑板中。
Measure the fluorescence intensity of the mixture (excitation at 322 nm and emission at 431 nm).测量混合物的荧光强度（激发波长 322 nm，发射波长 431 nm）。
The maximum and minimum values of the fluorescence intensity are obtained, and the median value is defined as pKa.记录荧光强度的最大值和最小值，并定义中位值为 pKa。
3.3 Confocal Laser Scan Microscopy 共聚焦激光扫描显微镜
Culture human fibrosarcoma cells (HT-1080) in D-MEM/Ham’s F-12 medium in a CO2 incubator (see Note 14).在 CO2 培养箱中，用 D-MEM/Ham’s F-12 培养基培养人类纤维肉瘤细胞（HT-1080）（见 注释 14）。
Seed HT-1080 cells onto a 24-well black plate at 1 × 104 cells/well and culture overnight (see Note 15).将 HT-1080 细胞以 1 × 10^4 细胞/孔接种到 24 孔黑板中，并孵育过夜（见 注释 15）。
Remove the medium and add 80 μL of fresh medium and 20 μL of the lipoplex (DiD-labeled polycationic liposome and FITC-labeled siRNA complex) at the siRNA concentration of 2.5 nM.去除培养基，加入 80 μL 新鲜培养基和 20 μL 脂质体复合物（DiD 标记的多阳离子脂质体与 FITC 标记的 siRNA 复合物），siRNA 浓度为 2.5 nM。
Nine hours after the start of transfection, remove the medium and wash the liposomes with PBS 3 times (see Note 16).在转染开始后 9 小时，去除培养基并用 PBS 洗涤脂质体 3 次（见 注释 16）。
Add 200 μL of medium without FBS.加入 200 μL 不含 FBS 的培养基。
Add 15 μL of LysoTracker and incubate for 2 h at 37 °C (see Note 17).添加 15 μL 的 LysoTracker 并在 37 °C 下孵育 2 小时（见 注释 17）。
Wash the cells thrice with PBS containing 30 U/mL heparin.用含 30 U/mL 肝素的 PBS 洗涤细胞 3 次。
Add 200 μL of 4% paraformaldehyde/PBS and incubate for 1 h at 37 °C.加入 200 μL 的 4%多聚甲醛/PBS，在 37 °C 下孵育 1 小时。
Wash the cells with PBS 3 times and block the cells with 3% BSA-0.1% saponin-PBS at 37 °C for 30 min.用 PBS 洗涤细胞 3 次，并用 3% BSA-0.1%皂素-PBS 阻断细胞 30 分钟。
Remove the medium and add 100 μL of DAPI, 3% BSA, 0.1% saponin/PBS and incubate for 15 min at 37 °C for the nuclear staining.去除培养基，加入 100 μL 的 DAPI、3% BSA、0.1%皂素/PBS，孵育 15 分钟以进行核染色。
Wash the cells with PBS once and then add a few drops of PERMAFLUOR aqueous mounting medium.用 PBS 洗涤细胞一次，然后加入几滴 PERMAFLUOR 水溶性封装介质。
Cover with a cover glass and stand for 1 h in a dark place (see Note 18).盖上盖玻片，在黑暗处静置 1 小时（见 注释 18）。
The location of the DiD-labeled polycation liposomes and FITC-labeled siRNA is observed by using confocal laser scanning microscopy (see Notes 19 and 20).使用共聚焦激光扫描显微镜观察 DiD 标记的多阳离子脂质体和 FITC 标记的 siRNA 的位置（见 注释 19和20）。
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Gentle vortexing should be performed before use, because the lipids will form deposits in the chloroform during the storage. If the lipid solution will be stored for a long time, we recommend that the lipid should be solubilized again, because the chloroform will gradually become volatilized during storage. 使用前应轻柔涡旋，因脂质在氯仿中存储时会形成沉淀。如需长时间存储，建议重新溶解脂质，因氯仿会逐渐挥发。
Dispense the dissolved siRNA. Do not repeat freeze-thawing. Repeating the freeze-thawing will cause siRNA degradation.分装溶解的 siRNA，避免重复冻融，因重复冻融会导致 siRNA 降解。
To avoid siRNA degradation, all buffers should be prepared with RNase-free water.为避免 siRNA 降解，所有缓冲液应使用无 RNase 水制备。
Caution: FBS-containing medium is easily contaminated. So, pay attention to handling it. After its preparation, use the medium within a month.注意：含 FBS 的培养基易被污染，因此操作时要小心。制备后一个月内使用。
Paraformaldehyde is a toxic reagent. Wear a mask, gloves, and protective eyewear when weighing the paraformaldehyde. Weighing inside a hood is highly recommended. To avoid exposing coworkers to the paraformaldehyde, cover the weighing boat. In addition, transfer the weighed paraformaldehyde to a cylinder inside the hood. As paraformaldehyde is dissolved in water with difficulty, stirring in a heated water bath (60 °C) is recommended. Use the paraformaldehyde solution within a week.多聚甲醛为有毒试剂，称量时应佩戴口罩、手套和护目镜，建议在通风柜中称量。称量后应覆盖称量容器，转移到圆柱体中时也应在通风柜内。因多聚甲醛在水中难溶，推荐在 60 °C 的水浴中搅拌。应在一周内使用制备好的溶液。
BSA-containing solution is easy to foam. Intense vortexing is not recommended, and gentle vortexing is better. BSA-containing solution is easily contaminated. Preparing it when needed is better. Do not repeat freeze-thawing the solution.含 BSA 的溶液易产生泡沫，不建议强烈涡旋，轻柔涡旋更佳。该溶液易被污染，最好在需要时制备，避免重复冻融。
Do not add a large amount of tert-butyl alcohol and ensure that the flask is not too small to avoid breaking it, as the solvent volume will increase after freezing.不要添加大量叔丁醇，并确保烧瓶大小合适，以免破裂，因为冷冻后溶剂体积会增加。
Evaporate the chloroform completely. After the evaporation, the remaining tert-butyl alcohol is removed by freeze-drying. Tert-butyl alcohol can be trapped in the refrigerated part. However, chloroform cannot be trapped. Evaporated chloroform will move to the pump, which may cause pump trouble.完全蒸发氯仿。蒸发后，剩余的叔丁醇需通过冻干去除。叔丁醇可被冷藏部分捕获，但氯仿不能。蒸发的氯仿将进入泵中，可能导致泵故障。
After the preparation of the RNase-free water, dispense the water while avoiding contamination. Hydration water should be heated above the phase-transition temperature of lipids before use.制备无 RNase 水后，应避免污染进行分装。使用前，水应加热至超过脂质的相变温度。
Positively charged lipids may strongly bind to the negatively charged glass flask. Short-term sonication may dissolve the problem.正电荷脂质可能与负电荷玻璃烧瓶强烈结合，短时间超声波处理可解决此问题。
Extrusion should be performed above the phase-transition temperature of lipids to form liposomes. Therefore, the extruder should be in a heated water bath during the extrusion process. Of course, the lipid solution should be heated above the phase transition temperature before being put into the extruder.挤出应在脂质的相变温度以上进行，以形成脂质体。因此，挤出机应在加热水浴中操作，脂质溶液也应加热至相变温度以上再放入挤出机。
Do not heat above the melting temperature of siRNA (around 50 °C) to avoid dissociation.不要加热至超过 siRNA 的熔点（约 50 °C），以避免解离。
Use of RNase-free water is recommended for the measurement of liposome size and PDI because cationic liposomes tend to aggregate in buffers.建议使用无 RNase 水测量脂质体大小和 PDI，因为阳离子脂质体在缓冲液中易聚集。
Do not culture cells at the confluent state. Culturing cells at high density may decrease the cell growth ratio and liposomal uptake. The cells should be sub-confluent after passage.不要在细胞融合状态下培养细胞，高密度培养可能降低细胞生长比和脂质体摄取。细胞在传代后应处于亚融合状态。
Evenly dispersing the cells in the wells is important. The cell density in the well will change liposome uptake. Extremely high cell density may decrease liposomal uptake. To avoid this problem, using a microscope to check the cell density just after cell seeding is better.确保细胞在孔中的均匀分布非常重要。孔中的细胞密度会影响脂质体摄取。极高的细胞密度可能降低脂质体的摄取。为避免此问题，接种后最好用显微镜检查细胞密度。
During the washing step, do not dry the cells, as the cells will become detached from the well if the cells are dried.在洗涤步骤中，不要让细胞干燥，因细胞会从孔中脱落。
Weakly acidic amine selectively accumulates in low-pH organelles such as endosomes and lysosomes. Since LysoTracker is a weakly acid fluorescent probe, it selectively accumulates in the acidic organelles. In addition, LysoTracker can penetrate the cellular membrane. Fluorescent signals in the cytoplasm will decrease once the cells are incubated in the medium without the probe after the staining. Since LysoTracker stains only some of the lysosomes, quantitative analysis is difficult.弱酸性胺选择性积累在低 pH 的细胞器中，如内体和溶酶体。由于 LysoTracker 是弱酸性荧光探针，它选择性地积累在酸性细胞器中。此外，LysoTracker 能够穿透细胞膜。细胞在无探针培养基中孵育后，细胞质中的荧光信号会下降。由于 LysoTracker 只标记部分溶酶体，定量分析较为困难。
Notice: Do not allow any bubble to be trapped when placing the cover glass on the glass slide.注意：放置盖玻片时，请勿让气泡被困住。
If you do not observe the sample immediately, store it at 4 °C in the dark to avoid quenching.如果不立即观察样本，应在 4 °C 暗处存放以避免荧光淬灭。
If the intensity of the cell staining is not enough, increase the concentration of the cell staining solution or extend the incubation time.如果细胞染色强度不足，可以增加染色溶液的浓度或延长孵育时间。
