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发表于 2016-8-3 18:23:38
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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.
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发表于 2016-8-2 14:25:28
，如果很短时间就做了，估计影响会小一点。