Hi all,
I have some BMDMs from various genotypes which I have stimulated overnight with LPS+zvad and LPS+zvad+Necrostatin to study differences in necroptosis. Most protocols I have read have used propidium iodide (PI) as a vital dye. But, at the time of the experiment I did not have access to PI and had to fix my cells in 4 % PFA. The cells are currently sitting in 1x PBS in 4 C. Can anyone recommend a good staining protocol for PI for fluorescence microscopy?
Thanks
For PFA fixed cells, PI staining require few steps
1) Permeabilise the cells : either with 0.1% triton or 0.1% saponin
2) You have to give RNAse treatment with 100ug/ml for 1h
Then you can stain your cells with PI
The answer above me makes little sense. If the cells are fixed they will all incorporate PI, no matter if they're live or dead. You don't need detergents for that as well; we do it routinely. If you want to use PI as a measure of viability you can't fix your cells because fixing kills them. And the Rnase treatment mentioned above is for when you want to check for cell cycle stage, as RNA contaminants bind PI and give confusing results.
- Badges
- Science method
- Similar topics
- Biological Science
- Histology
- Staining