Fluo-4 AM (钙离子荧光探针, 2mM)
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产品编号 S1060
数量
¥ 467.00
产品包装:
25μl
产品简介
使用说明
说明书下载
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Fluo-4 AM是最常用的检测细胞内钙离子浓度的荧光探针之一。分子式为C51H50F2N2O23,分子量为1096.95。
Fluo-4 AM在Fluo-3 AM的基础上改进而成,其特点是加载更快并且在相同条件下荧光更加明亮。Fluo-4 AM将Fluo-3 AM结构中的氯原子替换成了氟原子,导致它的最大激发波长会向短波长处偏离12nm左右。这个波长更接近于氩激光器的波长488nm,所以用氩激光器激发时,Fluo-4的荧光强度会比Fluo-3强一倍。由于Fluo-4与钙离子的亲和力和Fluo-3相近,所以使用上和Fluo-3也基本相同,可以使用激光共聚焦显微镜、荧光酶标仪等荧光检测仪器来测定细胞内钙离子浓度的变化。Fluo-4 AM和Fluo-3 AM的结构式参考图1。

图1. Fluo-4 AM和Fluo-3 AM的结构式。
Fluo-4 AM是Fluo-4的乙酰甲酯衍生物,是一种可以穿透细胞膜的荧光染料。Fluo-4 AM的荧光非常弱,其荧光不会随钙离子浓度升高而增强。Fluo-4 AM进入细胞后可以被细胞内的酯酶剪切形成Fluo-4,从而被滞留在细胞内。Fluo-4可以和钙离子结合,结合钙离子后可以产生较强的荧光,最大激发波长为494nm,最大发射波长为516nm。实际检测时推荐使用的激发波长为488nm左右,发射波长为512-520nm。Fluo-4的激发光谱和发射光谱参考图2。

图2. Fluo-4的激发光谱和发射光谱
用于细胞内钙离子检测时,Fluo-4 AM的常用浓度为0.5-5μM。通常用含有0.5-5μM的Fluo-4 AM的适当溶液和细胞一起在20-37℃孵育10-60分钟进行荧光探针装载,随后适当洗涤,洗涤后可以考虑适当再孵育20-30分钟以确保Fluo-4 AM在细胞内充分转变成Fluo-4。
本Fluo-4 AM(钙离子荧光探针) 是配制于无水DMSO (anhydrous DMSO)中的储存母液,浓度为2mM。
包装清单:

产品编号 产品名称 包装
S1060 Fluo-4 AM(钙离子荧光探针,2mM) 25μl
说明书
1份

保存条件:
-20℃避光保存,6个月有效。
注意事项:
Fluo-4 AM遇水极易分解,建议第一次使用时,母液分装并密封保存,同时注意保持干燥。
Fluo-4 AM在4℃、冰浴等较低温度情况下会凝固而粘在离心管管底、管壁或管盖内,可以20-25℃水浴温育片刻至全部融解后使用。
探针的工作浓度、细胞量、孵育温度和时间等需要根据不同实验预先摸索。
荧光染料均存在淬灭问题,请尽量注意避光,以减缓荧光淬灭。
本产品仅限于专业人员的科学研究用,不得用于临床诊断或治疗,不得用于食品或药品,不得存放于普通住宅内。
为了您的安全和健康,请穿实验服并戴一次性手套操作。

使用说明:
1.取适量Fluo-4 AM母液,用PBS稀释至0.5-5μM的工作液。
注:工作液须即用即配,请勿反复冻融。
2.对于待检测的培养细胞,去除培养液,用PBS或HBSS洗三遍。
注:因为培养液中的血清含有酯酶会导致Fluo-4 AM分解为Fluo-4,而酚红会导致荧光背景增强。
3.加入Fluo-4 AM工作液,溶液量以能充分覆盖细胞为准。
4.20℃-37℃孵育10-60分钟进行荧光探针装载。
注:如果是首次实验不能确定孵育温度和时间,建议先尝试37℃孵育30分钟,观察荧光效果。如果细胞死亡较多,则适当缩短时间或降低温度;如果荧光强度太弱,则适当延长时间。
5.随后用PBS或HBSS洗涤3次,洗涤后可以考虑适当再孵育20-30分钟以确保Fluo-4 AM在细胞内完全转变成Fluo-4。
6.如有需要,可以使用适当药物来刺激细胞。
7.用激光共聚焦显微镜、荧光酶标仪、荧光分光光度计或流式细胞仪等荧光检测仪器检测Fluo-4的荧光,以确定细胞内钙离子浓度的变化。

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