本品为红色荧光标记的溶酶体探针，具有 577/590 nm 的最大激发/发射波长。本品以溶于无水 DMSO 的 1 mM 储存液形式提供。原料均采用美国进口原料。该探针是对活细胞中的酸性区室进行选择性染色的一类荧光染料，具有几大重要的特点：
1）选择性标记酸性细胞器；
2）纳摩尔级（nM）浓度即可有效标记活细胞；
3）具有多色探针提供，可根据情况对样品进行多标实验。结构上由一个荧光基团和相连的弱碱基构成，可自由穿过细胞膜，一般聚集在球形细胞器上，适用于观察溶酶体内部生物合成及相关发病机理。中性 pH 下仅仅发生部分质子化，因此该探针标记细胞器的原理可能与其完全质子化并滞留在细胞器膜上有关。
用途及描述 ：科研试剂，广泛应用于分子生物学，药理学等科研方面，严禁用于人体。
使用方法（仅供参考）
使用前，先将本品取出回温至室温，并对其进行简短离心使 DMSO 溶液集中于管底。最佳工作浓度需根据不同的实验要求、细胞类型、细胞或组织的膜通透性等进行优化。
1.工作液的配制
利用培养基或合适的缓冲液将 1 mM 储存液稀释至工作浓度，推荐工作液浓度为 50-75 nM；
【注意】：（1）为了降低探针加载过度可能引起的假阳性，建议在不影响染色效果的情况下尽量使用低浓度。
（2）若细胞在染色后于不含染料的培养基中孵育，会观察到荧光信号的衰减和细胞的空泡化现象。
2.染色
2.1 对于贴壁细胞
（1） 将细胞置于培养皿中的盖玻片上，加入合适培养基，使其爬片生长。
（2） 待细胞生长到合适丰度，吸除培养液，加入适量 37℃预热的含探针工作液。于生长状态下孵育 30 min～2 h（具体孵育时间需根据细胞类型而定）。
（3） 利用新鲜培养基替换上述染色液并在荧光显微镜（含合适滤片）下观察。若染色不够充分，建议增加染料浓度或延长染色时间。
2.2 对于悬浮细胞
（1） 离心，吸除上清。
（2） 利用 37℃预热的探针工作液重悬细胞，于生长状态下孵育 30 min～2 h（具体时间需根据细胞类型而定）。
（3） 离心，吸除染色液，加入新鲜培养液重悬细胞。
（4） 置于荧光镜（含合适滤片）下观察。若染色不够充分，建议增加染料浓度或加长染色时间。
注意：对于悬浮细胞，也可将细胞贴附于经 BD Cell-Tak 处理过的盖玻片上，然后使用类似于贴壁细胞的方法进行染色。
注意事项
1.我司产品为非无菌包装，若用于细胞培养，请提前做预处理，除去热原细菌，否则会导致染菌。
2.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。
This product is a red fluorescent lysosomal probe with maximum excitation/emission wavelengths at 577/590 nm. It is supplied as a 1 mM stock solution dissolved in anhydrous DMSO, and all raw materials are imported from the United States. As a fluorescent dye that selectively labels acidic compartments in live cells, it possesses several key advantages:
1.Specific labeling of acidic organelles;
2.Effective staining of live cells at nanomolar (nM) concentrations;
3.A series of multi-color probes are available to support multi-labeling experiments as needed.
Structurally composed of a fluorophore linked to a weak base, the probe freely penetrates cell membranes and accumulates in spherical organelles. It is suitable for investigating lysosomal biosynthesis and related pathogenic mechanisms. Only partial protonation occurs at neutral pH; therefore, its labeling mechanism relies on full protonation and retention within organelle membranes.
Application and Description
This is a research reagent widely used in molecular biology, pharmacology and other research fields. It is strictly prohibited for human use.
Protocol (For Reference Only)
Before use, equilibrate the product to room temperature and perform a brief centrifugation to collect all DMSO solution at the tube bottom. The optimal working concentration should be optimized based on experimental requirements, cell type, and membrane permeability of cells or tissues.
1. Preparation of Working Solution
Dilute the 1 mM stock solution with culture medium or suitable buffer to prepare the working solution. The recommended working concentration ranges from 50 to 75 nM.
Notes:
(1) To reduce false positive signals caused by probe overloading, use the lowest feasible concentration without compromising staining performance.
(2) Fluorescence attenuation and cellular vacuolation may be observed if stained cells are incubated in dye-free medium after labeling.
2. Staining Protocol
2.1 Adherent Cells
(1) Seed cells onto coverslips placed in culture dishes with appropriate medium for adherent growth.
(2) When cells reach optimal confluency, discard the medium and add pre-warmed probe working solution at 37 °C. Incubate under standard culture conditions for 30 min to 2 h (incubation time varies by cell line).
(3) Replace staining solution with fresh medium and observe under a fluorescence microscope equipped with matching filter sets. Increase dye concentration or extend incubation time if staining intensity is insufficient.
2.2 Suspension Cells
(1) Centrifuge the cell suspension and discard the supernatant.
(2) Resuspend the cell pellet in pre-warmed probe working solution at 37 °C, then incubate under standard culture conditions for 30 min to 2 h (incubation time varies by cell line).
(3) Centrifuge again, remove staining solution, and resuspend cells in fresh culture medium.
(4) Observe under a fluorescence microscope with appropriate filter sets. Increase dye concentration or extend incubation time if staining intensity is insufficient.
Note: Suspension cells can also be immobilized on BD Cell-Tak-coated coverslips and stained following the protocol for adherent cells.
Precautions
(1) This product is not sterilely packaged. For cell culture applications, pretreat the solution to remove pyrogens and bacteria in advance to avoid microbial contamination.
(2) Only partial product information is available for certain items. The company does not guarantee the authority of the provided data, which are for reference and academic research only.
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