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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