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，后用活性氧阳性对照或自己感兴趣的药
    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，先用活性氧阳性对照或自己感兴趣
    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用无血清培养液稀释DCFH-DA，使终浓
    度为10微摩尔/升。去除细胞培养液，加入适当体积稀释好的DCFH-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DCFH-DA不少于1毫升。37℃细胞培养箱内孵育20分
    钟。用无血清细胞培养液洗涤细胞三次，以充分去除未进入细胞内的DCFH-DA。通常活性氧阳性对照
    在刺激细胞20-30分钟后可以显著提高活性氧水平。
    收集细胞后装载探针：按照1:1000用无血清培养液稀释DCFH-DA，使终浓度为10微摩尔/升。细胞
    收集后悬浮于稀释好的DCFH-DA中，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。每隔3-5分钟颠倒混匀一下，使探针和细胞充分接触。用无血清细胞培养液洗涤细胞三次，以充
    分去除未进入细胞内的DCFH-DA。直接用活性氧阳性对照或自己感兴趣的药物刺激细胞，或把细胞等
    分成若干份后刺激细胞。通常活性氧阳性对照在刺激细胞20-30分钟后可以显著提高活性氧水平。
    说明：仅在阳性对照孔中加入Rosup作为阳性对照，其余孔不必加入Rosup。
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，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488nm激发波长，525nm发射波长，实时或逐时间点检测刺激前后荧光的强弱。DCF的荧光光谱和
    FITC非常相似，可以用FITC的参数设置检测DCF。DCF的激发光谱和发射光谱参考下图。
                        
4. 其它说明
    阳性对照可以按照1:1000的比例使用。例如装载好探针的细胞共1毫升，可以加入1微升的阳性对照
    刺激。通常刺激后20-30分钟内可以观察到非常显著的活性氧水平升高。对于不同的细胞，活性氧阳
    性对照的效果可能有较大的差别。如果在刺激后30分钟内观察不到活性氧的升高，可以适当提高活
    性氧阳性对照的浓度。如果活性氧升高得过快，可以适当降低活性氧阳性对照的浓度。
    另外，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稀
    释DCFH-DA，使装载探针时DCFH-DA的浓度为2-5微摩尔/升。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。
    活性氧阳性对照(Rosup)仅仅用于作为阳性对照的样品，并不是在每个样品中都需加入活性氧阳性对
    照。

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