操作方法
1.准备DiO,DiI,DiD,DiS或DiR膜染色溶液:
1.1制备DMSO或EtOH储备溶液:储备溶液应在DMSO或EtOH中以1-5mM制备。
注意:储备溶液的未使用部分应储存在-20 ℃。 避免反复冻/融循环。
1.2准备工作溶液:将储备溶液(步骤1.1)稀释到合适的缓冲液中,如无血清培养基,HBSS或PBS制备1至5μM的工作溶液。
注意:对于不同的细胞类型和/或实验条件,应根据经验确定工作溶液的浓度。 建议在至少超过十倍范围的浓度下进行测试。
2.将细胞染成悬浮液:
2.1在染料工作溶液中悬浮细胞密度为1×106 / mL(来自步骤1.2)。
2.2在37°C孵育2-20分钟。 孵育时间取决于细胞类型。 首先孵育20分钟,然后根据需要进行优化以获得均匀的标记。
2.3将标记的悬浮管以1000至1500rpm离心5分钟。
2.4取出上清液,轻轻地将细胞重新悬浮在预热(37°C)的生长培养基中。
2.5按步骤2.3和2.4洗涤两次。
3.染色贴壁细胞:
3.1在无菌玻璃盖玻片上培养贴壁细胞。
3.2从生长培养基中取出盖玻片,轻轻地排出多余的培养基。 将盖玻片放在湿度箱中。
3.3将100μL染料工作溶液(来自步骤1.2)吸移到盖玻片的角落,轻轻搅拌直至所有细胞都被覆盖。
3.4将盖玻片在37°C孵育2-20分钟。 孵育时间取决于细胞类型。 首先孵育20分钟,然后根据需要进行优化以获得均匀的标记。
3.5排出染料工作溶液,用生长培养基清洗盖玻片2-3次。每个洗涤循环用预热的生长培养基覆盖细胞,孵育5-10分钟后排出培养基。
4.显微镜检测:
4.1说明书中的表1总结了DiD,DiO,DiI,DiS和DiR滤波器组的选择。
4.2为了同时检测多种染料,可提供如下多波段滤波器组:
a)DiI和DiO = Omega XF52,Chroma 51004
b)DiI和DiD = Omega XF92,Chroma 51007
c)DiI,DiO和DiD = Omega XF93,Chroma 61005
5.流式细胞仪检测:
用DiO,DiI,DiD,DiS和DiR标记的细胞可分别使用常规FL1,FL2,FL3和FL4流式细胞仪检测通道进行分析。
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