DAF-FM DA (NO荧光探针)
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产品编号 S0019
数量
¥ 746.00
产品包装:
>100次
产品简介
使用说明
说明书下载
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DAF-FM DA即3-Amino,4-aminomethyl-2',7'-difluorescein, diacetate,也称DAF-FM diacetate或4-Amino,5-
aminomethyl-2',7'-difluorescein, diacetate。DAF-FM DA是最新一代用于一氧化氮定量检测的荧光探针,比以前比较常用的一氧化氮荧光检测探针DAF-2 diacetate有多方面的改进。首先,DAF-FM DA和DAF-2 diacetate相比,最后和一氧化氮反应形成的荧光产物受pH值的影响小,在pH值大于5.5时不受pH值的影响。其次,DAF-FM DA和DAF-2 diacetate相比,前者产生的荧光更加稳定,不容易淬灭,这样更加便于检测。另外,DAF-FM DA和DAF-2 diacetate相比,前者对一氧化氮的检测灵敏度更高,相同条件下检测灵敏度可以提高接近2倍,最低检测浓度可以达到3nM。

   DAF-FM DA可以穿过细胞膜(cell-permeable),进入细胞后可以被细胞内的酯酶催化形成不能穿过细胞膜的DAF-FM。DAF-FM本身仅有很弱的荧光,但在和一氧化氮反应后可以产生强烈荧光,激发波长为495nm,发射波长为515nm。DAF-FM DA检测一氧化氮的机制可以参考上图。任何可以检测fluorescein的仪器,包括荧光显微镜、激光共聚焦显微镜、流式细胞仪、荧光分光光度计或荧光酶标仪都可以用于该荧光探针的检测。右图为DAF-FM在不同浓度一氧化氮存在时的发射荧光扫描图谱(fluorescence emission spectra)。
DAF-FM DA的分子量为496.4,分子式为C25H18F2N2O7,HPLC分析纯度大于98%。
本DAF-FM DA为溶解于DMSO的淡黄色溶液,浓度为5mM。
  本荧光探针适合于检测细胞内的一氧化氮水平,可以进行实时检测。如果收集细胞后再装载探针,通常至少可以检测100个样品。

包装清单:
产品编号 产品名称 包装
S0019-1 DAF-FM DA(NO荧光探针)5mM 20μl
S0019-2 DAF-FM DA稀释液 50ml
说明书 1份

保存条件:
-20℃保存,DAF-FM DA需避光保存,一年有效。
注意事项:
BSA和酚红(phenol red)对本荧光探针的检测有干扰,需避免。
第一次使用时请分装成小包装后-20℃保存,以避免反复冻融。
荧光探针应随用随配。配制好的探针工作液应立即使用,不能以后再用。
DAF-FM DA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。
      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. 装载探针
对于刺激时间较短(通常为2小时以内)的细胞,先装载探针,后用适当的阳性对照及自己感兴趣的药物刺激细胞。对于细胞刺激时间较长(通常为6小时以上)的细胞,先用适当的阳性对照及自己感兴趣的药物刺激细胞,后装载探针。
原位装载探针:本方法仅适用于贴壁培养细胞。按照1:1000比例,用本试剂盒提供的DAF-FM DA稀释液稀释DAF-FM DA,使终浓度为5微摩尔/升。去除细胞培养液,加入适当体积稀释好的DAF-FM DA。加入的体积以能充分盖住细胞为宜,通常对于六孔板的一个孔加入稀释好的DAF-FM DA的体积为1毫升。37℃细胞培养箱内孵育20分钟。用PBS(pH7.4)洗涤细胞三次,以充分去除未进入细胞内的DAF-FM DA。
收集细胞后装载探针:按照1:1000比例,用本试剂盒提供的DAF-FM DA稀释液稀释DAF-FM DA,使终浓度为5微摩尔/升。细胞收集后,用稀释好的DAF-FM DA重悬细胞,细胞浓度为一百万至二千万/毫升,37℃细胞培养箱内孵育20分钟。上述操作可以在离心管内进行。每隔3-5分钟颠倒混匀一下,使探针和细胞充分接触。用PBS(pH7.4)洗涤细胞三次,以充分去除未进入细胞内的DAF-FM DA。直接用适当的阳性对照或自己感兴趣的药物刺激细胞,或把细胞等分成若干份后再刺激细胞。
2. 检测
对于原位装载探针的样品可以用激光共聚焦显微镜直接观察(用普通的荧光显微镜观察效果相对较差),或收集细胞后用荧光分光光度计、荧光酶标仪或流式细胞仪检测。对于收集细胞后装载探针的样品可以用荧光分光光度计、荧光酶标仪或流式细胞仪检测,用激光共聚焦显微镜直接观察也可以。
3. 参数设置
使用495nm激发波长,515nm发射波长,实时、逐时间点或单时间点检测刺激前后荧光的强弱。DAF-FM和 一氧化氮反应产物的荧光光谱和fluorescein非常相似,可以用检测fluorescein的参数设置进行检测,用检测FITC的参数设置进行检测也可以。
4. 其它说明
上述推荐的DAF-FM DA的工作浓度为5微摩尔/升,对于某些细胞,如果发现没有刺激的阴性对照细胞荧光也比较强,可以按照1:2000-1:5000的比例稀释DAF-FM DA,使装载探针时DAF-FM DA的浓度为1-2.5微摩尔/升。相反,如果发现用感兴趣的药物刺激后荧光较弱,可以把DAF-FM DA的工作浓度为调整为10微摩尔/升,以提高检测的灵敏度。另外,探针装载的时间也可以根据情况在15-60分钟内适当进行调整。
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