SYTO9 是一种可以与核酸结合并发出明亮绿色荧光的细胞渗透性荧光染料。其具有细胞通透性，与核酸结合后量子产率显著提高。SYTO 9 可用于荧光显微镜、流式细胞术分析和 PCR 等，其PCR 抑制率低、信噪比高、灵敏度高，是 SYBR Green Ⅰ的理想替代染料。我司 SYTO9 绿色荧光核酸染料以高纯度粉末形式提供，可以根据使用浓度配制使用，操作灵活，用途广泛。
产品用途：
科研试剂，广泛应用于细胞生物学、分子生物学、药理学等科研方面，严禁用于人体。SYTO9是一种绿色荧光核酸染料，可用于：
1. 细胞染色：SYTO9 具有细胞膜渗透性，能够很好地渗透进入原核和真核细胞膜，广泛与细胞核、细胞质及线粒体内的 DNA 和 RNA 结合发出明亮的绿色荧光；
2. 细菌活死染色：SYTO 9 常与碘化丙啶（PI）联合使用，用于区分活细菌和死细菌，评估细菌的存活率或药物对细菌的影响；
3. RT-PCR、DNA 熔解曲线分析及环介导恒温扩增技术（LAMP）：SYTO 9 作为一款灵敏的DNA 结合染料，可以替代 SYBR Green Ⅰ用于 RT-PCR 等扩增实验中。
使用方法：（来自公开文献，仅供参考）
1. 储液配制：以 1mg 产品为例，加入 655μL DMSO，充分溶解，制备成 5mM 母液。溶解后的母液建议分装，避光储存于-20℃，可稳定保存 1 年；
2. SYTO 9 染色浓度和时间建议会根据不同的样本类型进行调整，推荐染色条件如下：
表 1. SYTO9 染色不同细胞的建议工作浓度和孵育条件
| 细胞类型 | SYTO 9 浓度 | 孵育条件 |
|---|
| 细菌 | 50nM~20μM | 涡旋混匀，孵育 1~30min | | 真核细胞 | 10nM~5μM | 孵育 10~120min | | 微阵列 | 50nM in TE buffer | 孵育 5min，清洗之后晾干 |
|
3. 细胞染色
（1）工作液配制：用 PBS 或培养基将 5mM 母液稀释至适当浓度（根据表 1 选择）；
（2）收集细胞，用 PBS 或培养基清洗 1-2 次；
（3）加入适量含 SYTO 9 的染色工作液（根据表 1 选择浓度及时间）；
（4）避光孵育后，直接进行荧光检测（激发 / 发射：~485/498 nm）或显微镜观察。
4. 活死细菌染色
（1）工作液配制：稀释 SYTO 9 和 PI，使其浓度分别为 33.4μM 和 400μM；
（2）取菌液 1 mL，离心收集，重悬于 PBS；
（3）加入 SYTO 9 和 PI 工作液各 50μL，使其终浓度分别为 1.67μM 和 20μM；
（4）室温避光孵育 15min，立即用荧光显微镜或流式细胞仪检测。
5. RT-PCR 与 DNA 熔解曲线分析
（1）按照常规 qPCR 体系配制反应液（引物、模板、dNTP、Taq 酶、缓冲液）；
（2）加入 SYTO 9 至终浓度 2μM（建议优化）；
（3）进行 PCR 扩增，在延伸阶段采集荧光信号；
（4）扩增结束后运行熔解曲线程序（如 60-95℃，每 0.5℃采集一次），分析 Tm 值。
6. 环介导恒温扩增技术（LAMP）
（1）配制 LAMP 反应混合液（引物、链置换 DNA 聚合酶、dNTP、模板、缓冲液）；
（2）加入 SYTO 9 至终浓度 0.5-10μM；
（3）恒温（如 63-65℃）反应 60min，每 30-60 秒采集一次荧光信号（Ex/Em=485/498nm）；
（4）根据荧光曲线判断扩增结果（阳性样品出现典型 S 型曲线）。
溶液配制：
【注意】
●首次开封后按单次用量分装于 PP/PE 管中，-20℃避光保存，避免反复冻融。取用前若余量较少，请快速离心数秒钟，使液体充分沉降。
●SYTO 9 易吸附玻璃材质，全程请使用塑料（PP/PE）器皿，避免使用玻璃容器。
●建议使用不含磷酸盐的缓冲液，以获得更好的染色效果。
●远离荧光淬灭剂：避免叠氮化物、>10mM EDTA 或含巯基的缓冲液（如 DTT、β- 巯基乙醇）。
●使用温和洗涤剂、热自来水、去离子水依次冲洗器皿数次，彻底去除残留荧光背景物质。
●本产品为非无菌包装。若用于细胞培养等无菌实验，请提前进行除菌处理（如 0.22μm 滤膜过滤），防止污染。
●为了您的安全和健康，请穿实验服并戴一次性手套操作。
●部分产品我司仅能提供部分信息，我司不保证所提供信息的权威性，以上数据仅供参考交流研究之用。
SYTO 9 is a cell-permeable fluorescent dye that binds nucleic acids and emits bright green fluorescence. It exhibits excellent cell permeability, and its quantum yield increases dramatically upon binding to nucleic acids. SYTO 9 is compatible with fluorescence microscopy, flow cytometry, PCR and other assays. Featuring low PCR inhibition, high signal-to-noise ratio and superior sensitivity, it serves as an ideal alternative to SYBR Green I. Our SYTO 9 green fluorescent nucleic acid dye is supplied as high-purity powder. Users can prepare working solutions at custom concentrations for flexible operation and broad application scope.
Product Applications
This product is a research reagent widely used in cell biology, molecular biology, pharmacology and other scientific research fields. It is strictly prohibited for human clinical use. As a green fluorescent nucleic acid stain, SYTO 9 can be applied in the following scenarios:
1. Cell staining: SYTO 9 possesses membrane permeability, readily penetrating the membranes of prokaryotic and eukaryotic cells. It broadly binds DNA and RNA within the nucleus, cytoplasm and mitochondria, generating intense green fluorescence.
2. Live/dead bacterial staining: SYTO 9 is commonly combined with propidium iodide (PI) to distinguish live and dead bacteria, enabling evaluation of bacterial viability or the antibacterial effects of compounds.
3. RT-PCR, DNA melting curve analysis and Loop-Mediated Isothermal Amplification (LAMP): As a sensitive DNA-binding dye, SYTO 9 can replace SYBR Green I for PCR-based amplification experiments such as RT-PCR.
Protocols (Adapted from published literature for reference only)
1. Stock Solution Preparation
Take 1 mg powder as an example: add 655 μL DMSO and dissolve completely to prepare a 5 mM stock solution. Aliquot the finished stock, store at −20 °C protected from light, and it remains stable for 1 year.
2. Recommended Staining Concentrations & Incubation Conditions
Staining parameters of SYTO 9 vary across sample types. Recommended protocols are listed in Table 1.
Table 1 Recommended working concentrations and incubation conditions of SYTO 9 for different cell samples
| Cell Type | SYTO 9 Concentration | Incubation Conditions |
|---|
| Bacteria | 50 nM ~ 20 μM | Vortex thoroughly, incubate for 1–30 min | | Eukaryotic cells | 10 nM ~ 5 μM | Incubate for 10–120 min | | Microarray | 50 nM in TE buffer | Incubate for 5 min, wash and air-dry afterward |
|
3. Cell Staining
(1) Prepare working solution: Dilute the 5 mM stock solution to the target concentration with PBS or cell culture medium (refer to Table 1 for parameters).
(2) Harvest cells and rinse 1–2 times with PBS or culture medium.
(3) Add an appropriate volume of SYTO 9 staining working solution (adjust concentration and incubation time per Table 1).
(4) Incubate in the dark, then perform fluorescence detection (Ex/Em: ~485/498 nm) or microscopic observation directly.
4. Live/Dead Bacterial Staining
(1) Prepare mixed working solution: Dilute SYTO 9 and PI to final concentrations of 33.4 μM and 400 μM, respectively.
(2) Collect 1 mL bacterial suspension by centrifugation, then resuspend the pellet in PBS.
(3) Add 50 μL each of SYTO 9 and PI working solution to reach final concentrations of 1.67 μM (SYTO 9) and 20 μM (PI).
(4) Incubate for 15 min at room temperature in the dark, then immediately analyze via fluorescence microscopy or flow cytometry.
5. RT-PCR and DNA Melting Curve Analysis
(1) Prepare standard qPCR reaction mixture containing primers, template, dNTPs, Taq DNA polymerase and buffer.
(2) Add SYTO 9 to a final concentration of 2 μM (optimization is recommended).
(3) Perform PCR amplification, and collect fluorescence signals during the extension step.
(4) Run melting curve program after amplification (e.g., 60–95 °C, signal acquisition every 0.5 °C) to analyze Tm values.
6. Loop-Mediated Isothermal Amplification (LAMP)
(1) Prepare LAMP reaction mixture containing primers, strand-displacement DNA polymerase, dNTPs, template and buffer.
(2) Add SYTO 9 to a final concentration of 0.5–10 μM.
(3) Carry out isothermal reaction at constant temperature (e.g., 63–65 °C) for 60 min, collect fluorescence signals every 30–60 s (Ex/Em = 485/498 nm).
(4) Judge amplification results based on fluorescence curves: positive samples generate characteristic S-shaped curves.
Solution Preparation
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Important Notes
• After initial opening, aliquot the reagent into PP/PE tubes according to single-use volume, store at −20 °C protected from light, and avoid repeated freeze-thaw cycles. If the remaining volume is low before use, perform a brief quick spin for several seconds to fully settle the liquid.
• SYTO 9 readily adsorbs to glass surfaces; use plastic (PP/PE) labware throughout the experiment and avoid glass containers.
• Phosphate-free buffers are recommended for optimal staining performance.
• Keep away from fluorescence quenchers: azides, EDTA at concentrations above 10 mM, and thiol-containing buffers (e.g., DTT, β-mercaptoethanol).
• Rinse labware sequentially with mild detergent, hot tap water, and deionized water several times to thoroughly eliminate residual fluorescent background contaminants.
• This product is non-sterile. For sterile experiments such as cell culture, pre-sterilize the reagent via filtration through a 0.22 μm membrane to prevent contamination.
• Wear a lab coat and disposable gloves during operation for personal safety and health protection.
• Our company only provides partial information for some products and does not warrant the authority of the provided data. All data above are for reference and research communication only.
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