贴壁细胞测凋亡做AV-PI实验。

zhouweiwei

由于最近试验不顺利，使用AV-PI检测贴壁细胞的凋亡率，FITC的阳性总是很高，所以查了查文献看有没有好方法，招到了一篇文献说现将AV-FITC加入培养基，随后用刮刀将细胞刮下，离心随后加入PI大家看这种方法是否靠谱呢？

原文摘录如下：Surface exposure of PS by apoptotic cells was measured by adding annexin V-FITC to the culture medium in a final concentration of 3 mg/ml. The cells were subsequently incubated for 3 min at room temperature. Detached cells were collected with the culture supernatant, pelleted by centrifugation and washed twice with culture medium in order to remove excess of annexin V-FITC. The adherent cells were rinsed twice with medium before harvesting. Cells were harvested by mechanical scraping with a rubber police man. Detached and adherent cells were finally pooled and resuspended in culture medium to a final concentration of 1×10⁶ cells/ml. Prior to flow cytometric analysis, PI was added to a final concentration of  5 mg/ml.

https://www.flowcyto.cn/bbs/forum.php?mod=attachment&aid=MTE0ODR8MWUyMzAzNzd8MTcxNjEyNDMwOXwwfA%3D%3D

niwanmao

贴壁细胞的AV、PI染色确实很难，你真的很用心，找到了这个有意思的方法。

看图形十分靠谱，但是Annexin V的结合需要含钙离子的缓冲液，这个步骤里面似乎没写，你可以在第一步里面将细胞培养基换成试剂盒中的缓冲液，结合3分钟，然后继续后面的步骤，或许可行。

希望这篇文章不是数据造假。

ps654321

长了见识

ddn111

这方法好像行不通：
1、AnnexinV -FITC加到培养基中，还用到3mg/ml的浓度，这大的量，国内哪个老板受得了？
2、看文章中的图右下区域AV-/ PI+的细胞那么多，难道不是刮死的细胞？

niwanmao

@ddn111  确实说到重点了，这个量还真是大啊。  不过下图的AV-PI+稍高一点我倒觉得问题不大，因为贴壁细胞无论哪种方法都无法避免，况且结果主要是要看AV+PI-的凋亡细胞。

轩辕河图

建议考虑一下细胞损伤，个人认为先染色后刮取细胞适用于一些结合比较弱或者浓度比较低的情况，然而在刮取过程中会不可避免的造成损伤，磷脂酰丝氨酸假阳性结合还是会有的。