7-AAD染色胞内细胞因子

浪漫纷飞

用7-AAD表染，洗涤后，再固定、打孔，圈门时仍可以用7-AAD-细胞代表活细胞的吧。毕竟7-AAD是固定打孔之前染的，如果染上的就是固定、打孔之前就死的细胞啊。

niwanmao

浪漫纷飞 发表于 2016-8-3 16:20
为什么呢？多洗两遍，去掉剩下的7-AAD，活细胞染不上的啊。

我明白你的设想，就是先染7-AAD后，洗涤几次，再进行固定破膜，然而，现实很骨感。引用1994年一篇文献的测试（Schmid I, Uittenbogaart CH, Keld B, Giorgi JV. A rapid method for measuring apoptosis and dual-color immunofluorescence by single laser flow cytometry. J Immunol Methods. 1994 Apr 15;170(2):145-57.）：

2.3. Staining using 7-AAD and flow cytometry
For staining of apoptotic and dead ceils with 7-AAD alone, cells were incubated with 20 ug/ml of 7-AAD in PBSAz for 20 min at 4°C protected from light; then the cells were analyzed on the flow cytometer in their staining solution.
When the samples were subsequently fixed in paraformaldehyde (PF) solution (Jackson et al., 1986), they were centrifuged once, the supernatant was removed and a 1% PF solution in PBS containing 20 ug/ml of actinomycin D (AD, Boehringer Mannheim, Indianapolis, IN) (PF/AD) was added to the cell pellet followed by incubation overnight. The samples were submitted to flow cytometric analysis in the PF/AD solution. Due to the removal of fluorescent 7-AAD out of solution of the samples that were then fixed in PF/AD, the staining intensity in the fixed sampies was decreased compared to the unfixed samples. Cells were kept up to 5 days in the fixative without detrimental effects on the ability to discriminate live from apoptotic, and from dead cells.

niwanmao

7-AAD不适合固定破膜染色时区分死活，不管什么时候染。如果要用得用一些商业化的专用于固定过细胞的死活鉴别染料。

zal86

同上，7-AAD 和PI都不适合固定后分析

浪漫纷飞

zal86 发表于 2016-8-2 16:50
同上，7-AAD 和PI都不适合固定后分析

为什么呢?

浪漫纷飞

zal86 发表于 2016-8-2 16:50
同上，7-AAD 和PI都不适合固定后分析

为什么呢？多洗两遍，去掉剩下的7-AAD，活细胞染不上的啊。

浪漫纷飞

niwanmao 发表于 2016-8-3 18:23
我明白你的设想，就是先染7-AAD后，洗涤几次，再进行固定破膜，然而，现实很骨感。引用1994年一篇文献的测 ...

非常感谢，非常专业的解答。不过他染色是不是太久了点？个人理解 overninght，7-AAD自然会乱串了，如果很短时间就做了，估计影响会小一点。