Lysosome tracer系列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，该类探针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。
Lysosome tracer 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。Lysosome tracer 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。本品Lysosome tracer Green DND-26 为绿色荧光标记的溶酶体探针，具有 504/511nm 的最大激发/发射波长。本品以溶于无水 DMSO 的 1mM 储存液形式提供。
注意事项：
1. 溶解性是在室温下测定的，如果温度过低，可能会影响其溶解性。
2. 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。
3. 本品仅供科研使用，勿做其他用途。
4. 为了您的安全和健康，请全程穿实验服并戴一次性手套操作。
使用说明：
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 1mM 储存液稀释至工作浓度，推荐工作液浓度为 50-75nM；
【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 30min～2h（具体孵育时间需根据细胞类型而定）。
【注意】：对 Lysosome tracer Green DND-26 内化过程的动力学研究表明，活细胞摄取此染料仅需数秒即可。
缺点在于此探针可能引起溶酶体产生“碱性效应 Alkalizing effect”，也就是说过长孵育时间会诱导溶酶体pH值提高。建议仅当该探针于 37℃孵育细胞 1-5min 才可用作 pH 指示剂。
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 30min～2h（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。
Lysosome tracer Series probes are a type of fluorescent dye that selectively stain acidic compartments in living cells. These probes have several important characteristics, including:
1) selective labeling of acidic organelles;
2) A nanomolar (nM) concentration can effectively label live cells;
3) Equipped with multi-color probes, multiple standard experiments can be conducted on the sample according to the situation.
Lysosome tracer Structurally composed of a fluorescent group and a connected weak base group, it can freely penetrate the cell membrane and generally aggregate on spherical organelles, suitable for observing the biosynthesis and related pathogenesis inside lysosomes. Lysosome tracer undergoes only partial protonation at neutral pH, so the principle of labeling organelles with this probe may be related to its complete protonation and retention on the organelle membrane. This product Lysosome tracer Green DND-26 is a lysosome probe labeled with green fluorescence, with a maximum excitation/emission wavelength of 504/511nm. This product is provided in the form of a 1mM storage solution dissolved in anhydrous DMSO.
Precautions：
1. Solubility is measured at room temperature, and if the temperature is too low, it may affect its solubility.
2. Our products are non sterile packaging. If used for cell culture, please perform pre-treatment in advance to remove pyrogenic bacteria, otherwise it may cause bacterial contamination.
3. This product is for scientific research purposes only and should not be used for any other purposes.
4. For your safety and health, please wear lab clothes and disposable gloves throughout the operation.
Instructions for Use：
Before use, take out the product and warm it back to room temperature, then perform a brief centrifugation to concentrate the DMSO solution at the bottom of the tube. The optimal working concentration needs to be optimized according to different experimental requirements, cell types, membrane permeability of cells or tissues, etc.
1. Preparation of working fluid
Dilute 1mM storage solution to the working concentration using culture medium or appropriate buffer, with a recommended working solution concentration of 50-75nM;
[Attention]:
1) To reduce the possibility of false positives caused by excessive probe loading, it is recommended to use low concentrations as much as possible without affecting the staining effect.
2) If cells are incubated in a dye free medium after staining, attenuation of fluorescence signals and vacuolization of cells will be observed.
2. Staining
2.1 For adherent cells
1) Place the cells on a cover glass in a culture dish, add suitable culture medium, and allow them to grow on the slide.
2) Once the cells have grown to an appropriate abundance, remove the culture medium and add an appropriate amount of probe containing working solution preheated at 37 ℃. Incubate for 30 minutes to 2 hours in a growing state (specific incubation time depends on cell type).
[Note]: Dynamics studies on the internalization process of Lysosome tracer Green DND-26 indicate that live cells only need a few seconds to uptake this dye.
The disadvantage is that this probe may cause lysosomes to produce an "alkaline effect", which means that prolonged incubation time can induce an increase in lysosome pH. It is recommended that the probe can only be used as a pH indicator when cells are incubated at 37 ℃ for 1-5 minutes.
3) Replace the above staining solution with fresh culture medium and observe under a fluorescence microscope (with appropriate filter). If the dyeing is not sufficient, it is recommended to increase the dye concentration or extend the dyeing time.
2.2 For suspended cells
1) Centrifuge and remove the supernatant.
2) Resuspend cells using probe working solution preheated at 37 ℃ and incubate in a growing state for 30 minutes to 2 hours (specific time depends on cell type).
3) Centrifuge, remove the staining solution, and resuspend the cells in fresh culture medium.
4) Observe under a fluorescent microscope. If the dyeing is not sufficient, it is recommended to increase the dye concentration or prolong the dyeing time.
[Note]: For suspended cells, they can also be attached to cover glass treated with BD Cell Tag, and then stained using a method similar to that of adherent cells.
ZJ26F0838591
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