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产品描述
Calcein-AM是一种对活细胞进行荧光标记的细胞染色试剂，发绿色荧光（Ex=490 nm，Em=515 nm）。 因其在传统的Calcein（钙黄绿素）基础上引入乙酰甲氧基甲酯（AM）基团，增加了疏水性，使其能够轻易穿透活细胞膜。一旦进入细胞后，Calcein-AM（本身不发荧光）被细胞内的酯酶剪切形成膜非渗透性的极性分子Calcein，从而被滞留在细胞内并发出强绿色荧光。与其它同类试剂（如BCECF-AM和CFDA）相比，由于Calcein, AM细胞毒性极低，是最适合用于活细胞染色的荧光探针，而且不会抑制任何的细胞功能，如增殖和淋巴球的趋化性。
由于死细胞缺乏酯酶，Calcein, AM仅用于对活细胞的细胞生存能力测试和短期标记。作为核染色染料的碘化丙啶不能穿过活细胞的细胞膜，它穿过死细胞膜的无序区域而到达细胞核，并嵌入细胞的DNA双螺旋从而产生红色荧光(激发：535 nm，发射：617 nm)，因此PI仅对死细胞染色。由于Calcein和PI-DNA都可被490 nm激发，因此可用荧光显微镜同时观察活细胞和死细胞。而用545 nm激发，仅可观察到死细胞。根据以上特点，Calcein, AM和PI经常被结合用来作为活细胞和死细胞的双重染色。由于不同细胞系的最佳染色条件不同，我们建议个别确定 Calcein, AM 和 PI的合适浓度。
本品为粉末形式提供的Calcein-AM，超纯级别（≥95%），可与碘化丙啶（PI）（Cat No. 40710ES03）联合使用，用于活细胞和死细胞的同时检测。或者直接购买翌圣提供的Calcein-AM/PI活细胞/死细胞双染试剂盒（Cat No. 40747ES76）。
产品性质
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中文名称（Chinese synonym） |
3'-O-乙酰胺-2',7'-二(羧乙基)-4或5-羧基荧光素，二乙酰甲酯；钙黄绿素乙酰甲酯； |
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英文名称（English synonym） |
3',6'-Di(O-acetyl)-4',5'-bis[N,N-bis(carboxymethyl)aminomethyl]fluorescein, tetraacetoxymethyl ester |
|
CAS号（ CAS NO） |
148504-34-1 |
|
分子式（Formula） |
C46H46N2O23 |
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分子量（Mol. Wt.） |
994.86 |
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纯度（Purity） |
≥95%（HPLC） |
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Ex/ Em（nm） |
490/515 |
|
结构式 |
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运输与保存方法
冰袋（wet ice）运输。-20℃干燥保存，避免直接暴露于强光下，有效期一年。
注意事项
1）对于微量试剂，开封前，请稍微离心一下，以保证粉末落入管底。
2）由于Calcein-AM对湿度非常敏感，本粉末保存的过程中一定要保持干燥。对于配制好的Calcein-AM储存液需要分装冻存，且必须紧紧密封盖子，干燥保存。Calcein-AM工作液必须现配现用。
3）为了您的安全和健康，请穿实验服并戴一次性手套操作。
4）本产品仅作科研用途！
使用方法
1. 储存液的配制
本品是以粉末形式提供的，根据工作液的浓度将其配制成1000×的储存液，储存液配制范围可为1～50 mM。例如使用的工作液为5 μM，那么可配制5 mM的储存液，也就是向50μg Calcein, AM（Mw：994.86）内加入10 μL 细胞培养级别的DMSO（CAS NO. 60313ES60）即可得到所需浓度的储存液。
2. 染色步骤
对于大部分细胞，钙黄绿素Calcein, AM的工作液浓度为2-5 μM。由于不同细胞系的最佳染色条件不同，初次实验建议做梯度实验，以确定 Calcein-AM的最适浓度。梯度筛选的原则为使用最低的探针浓度得到最好的荧光结果。
1）使用时，取一管适量的Calcein, AM储存液（1000×），用PBS（或Hanks和Hepes）缓冲液将其稀释成相应浓度的染色工作液。
【注】：有时候可添加一定量的非离子表面活性剂如Pluronic F127（Cat No. 60318ES60）到Calcein AM储存液内来增强其水溶性。制备染色工作液前，取一管需要用的Calcein AM储存液内加入等体积的20% Pluronic F127，使Pluronic F127的终浓度为0.02%。含Pluronic F127的Calcein AM溶液不可长期保存，现配现用。
2）对于贴壁细胞，先用胰酶-EDTA消化细胞，离心收集细胞（1000 rpm，3 min）。对于悬浮细胞，直接离心收集细胞。
3）去上清，用PBS（或者其他缓冲液）充分清洗细胞2~3次，以充分去除残留的酯酶活性。
4）用1/10细胞培养基体积的Calcein, AM染色工作液重悬细胞，37℃培养细胞15～30分钟。
【注】：如果细胞本身含有有机阴离子转运体，需加入丙磺舒（probenecid，1-2.5mM）或者苯磺唑酮（sulfinpyrazone，0.1-0.25mM）到孵育体系内以降低去酯化染料calcein泄露到胞外。
5）用PBS（或者其他缓冲液）洗涤细胞两次去除多余的染料。
【注】：如有必要，使用含有阴离子转运抑制剂的缓冲液来进行细胞清洗】
6）用含490 nm激发波长，515 nm发射波长的滤光片的荧光显微镜观察细胞。
相关产品
|
40719ES60 |
Calcein, AM, Ultrapure Grade 钙黄绿素，超纯级 |
2×50 μg |
|
40747ES76 |
Calcein-AM/PI Double Stain Kit Calcein-AM/PI活细胞/死细胞双染试剂盒 |
500 T |
|
40747ES80 |
Calcein-AM/PI Double Stain Kit Calcein-AM/PI活细胞/死细胞双染试剂盒 |
1000 T |
|
60318ES60 |
Pluronic® F-127 |
100 mg |
|
60313ES60 |
DMSO 二甲基亚砜，细胞培养级 |
100 mL |
HB221102
Q：这些探针染细胞，有没有细胞特异性？会不会 293 细胞不好染，MSC 好染？
A：由于这些染料是亲脂性染料，是通过插入到脂质膜中进而被激光激发观察，对于动植物细胞没有特异性。
Q：这些探针荧光探针装载完多久后观察？
A：为减少各种可能的误差，尽量缩短探针装载后到仪器测定所用的时间(刺激时间除外)。
Q：这些探针染色细胞后，对细胞有毒性吗？
A：荧光探针类产品对活细胞都会有一定的毒性，因此建议在染色处理完成后应尽快完成检测和观察。
Q：为什么染色后发现荧光颜色较浅，应怎么操作进行改进？
A：可以尝试增加染色的时间和浓度。
Q：要看几个小时过程中活细胞的形态变化，应该怎么选？
A：通常使用亲脂性花青染料，如 DiI，DiO，DiD 或 DiR。
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