As Caspase 3 assay is not applicable in S. cerevisiae, so what would be a good method to measure apoptosis/PCD in yeast cells?
We would be interested to have a simple method (maybe simpler than staining with tagged Annexin V?).
Thanks!
Do you have access to the deletion and GFP fusion libraries? If so then a CFU assay with the MCA1/AIF1/YSP1 deletion mutants is a good way. If you have GFP library access I would go for NUC1-GFP nuclear translocation assay for apoptosis with a counter-assay using NHP6A-GFP as a marker necrosis. You can also look for mitochondrial fragmentation, or if, you want to get more involved, cytochrome-C release by western blot. Bear in mid there are numerous "sub-forms" of yeast apoptosis, it all depends on what you are trying to achieve.
Annexin V/propidium iodide (PI) costaining was used to quantify externalization of phosphatidylserine, an early apoptotic event, and membrane permeabilization, which is indicative for necrotic death. This staining allows a discrimination between early apoptotic (Annexin V positive, PI negative), late apoptotic (Annexin V positive, PI positive), and necrotic (Annexin V negative, PI positive) death.
http://www.sciencedirect.com/science/article/pii/S1097276506008951
Maybe you should try some dyes like Methylene Blue? It is usually inexpensive and the methods are fast and simple. Check the link.
Good luck!
http://bkyeast.wordpress.com/2013/01/26/dyeing-yeast-cells-life-vs-death/
Thanks a lot!
We do use Methylene Blue, but as I would like to quantify apoptotic cells (and distinguish from necrotic cells), I will try the Annexin V/PI assay.
Methylene blue only permit you to make nuclei more visible and distingue between viable and death cells .The staining allows a discrimination between early apoptotic (Annexin V positive, PI negative), late apoptotic (Annexin V positive, PI positive), and necrotic (Annexin V negative, PI positive) death.
Good lock
It is possible to analyze chromatin condensation by staining with DAPI and DNA fragmentation by TUNEL test. This latter is easiest and faster especially if you have a flowcytometer. Also the evaluation of cell sensitivity to Cycloheximide might be a good apoptotic marker.
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