产品描述

LysoTracker^®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。LysoTracker^®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。Lysotracker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LysoTracker^® Green DND-26为绿色荧光标记的溶酶体探针，具有504/511 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。

产品性质

运输和保存方法

室温运输。-20℃避光保存，避免反复冻融。有效期2年。

注意事项

1）为了您的安全和健康，请穿实验服并戴一次性手套操作。

2）本产品仅作科研用途！

使用方法

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】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）工作液现配现用。

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）。

【注】：对Lysotracker 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1-5 min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】：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。

HB221025

3. 染色完到上机检测这一段时间过长，并且没有避光处理。建议避光染色，染色完立即检测荧光信号。

Q：在活细胞状态下，用 50 M 探针染色 50 min，然后发现除了溶酶体，细胞质中也有荧光信号， 这主要是什么原因导致？

A：大致有一下 2 个原因：

1. 溶酶体探针浓度过大，建议适当降低浓度；
2. 溶酶体探针染色时间过长，建议适当缩短时间。

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Q：染色时，细胞的状态？

A：这四种探针是对活细胞中的酸性区室进行染色。所以必须要在活细胞状态下染色。

Q：染色完成后，细胞是否能固定后再检测荧光信号？

A：固定后会破坏溶酶体的酸性环境从而会造成荧光减弱甚至消失的情况，染色前后均不建议固定。

Q：可以和二抗一起孵育吗？

A：不可以，孵育二抗必须要透化处理，而透化会破坏溶酶体的酸性环境，影响染色效果。

Q：这个探针可以染自噬溶酶体吗？可以染色其他酸性细胞器吗？

A：自噬溶酶体和正常的溶酶体在形态结构上有较大差异，该探针主要是针对常规的溶酶体染色，对于自噬溶酶体的染色效果不是很确定，可以参考一些使用过该产品发表的文献资料确定可以染色其他酸性细胞器，建议极低浓度下才能优先选择染色酸性溶酶体。

Q：植物细胞和组织适用吗？

A：理论上可以，一般植物细胞或者组织要制成原生质体。

Q：染色后可以放置一夜，再荧光检测吗？

A：建议染色完立即检测荧光，时间过长，荧光信号会逐渐减弱。

Q：用细胞培养基配成工作液以后稳定吗？可以存放多久？

A：工作液是需要现配现用的，不建议保存。

Q：检测仪器？

A：荧光显微镜 共聚焦显微镜 酶标仪 流式细胞仪。

Q：如何做到同时染色溶酶体和细胞核的？

A：建议用Hoechst 33258/ Hoechst 33342 染色细胞核，这个染料可以直接染活细胞不需要固定操作。

Q：保质期多久？

A：-20℃避光保存，半年有效。

Q：荧光信号较弱？

A：大致有 3 个原因：

1.溶酶体探针浓度过低，建议适当增大浓度；

2.染色时间过短，建议延长染色时间；

3.染色完之后，放置过长时间才检测信号；建议染色完立刻检测。

Q：没有荧光信号？

A：大致有 3 个原因：

1. 先用多聚甲醛 乙醛等固定细胞，破坏了溶酶体的酸性环境。
2. 染色完后，又用多聚甲醛固定细胞，或者透化细胞，破坏了溶酶体的酸性环境；染色前后均不建议固定细胞；