For many investigators the induction time of apoptosis is critical in establishing the importance of other putative apoptotic signals including cytochrome c release and mitochondrial membrane depolarization. Clontechs annexinV-EGFP technique detects early apoptosis and uses only one reagent to do so - protocols don't get any simpler than this one. The annexin V-EGFP is probably one of the most precise techniques available to examine a very early apoptotic event, that is, the time when apoptotic induction signals are telling the cell to "flip" phosphatidyl serine to the outer leaflet of the plasma membrane. This technique can be formatted for a 96 well assay and is extremely simple to perform. Even I can do this method over and over again without operator error!
I currently use Clontechs annexin V-EGFP kit for the detection of early apoptosis induced by redox modulation in the human B lymphoma cell line PW. The Clontech methodology has enabled me to pinpoint the induction time with great precision. Whereas propidium iodide (PI) staining alone indicated that apoptotic induction occurred at >72 hours after redox modulation with L-buthionine sulfoximine of PW cells, Clontechs AnnexinV-EGFP methodology has allowed me to define the true apoptosis induction time, at least 24 hours earlier, with great precision. This has allowed me to investigate cytochrome c release and MMP depolarization as "cause or consequence" in redox dependent PW cell apoptosis. At the outset the price of the kit may seems on the high side, but for the information generated using this technology, its well worth every penny.
Why choose GFP over FITC labeling? Well the GFP fluorescent signal has one of the highest signal to noise ratios. Indeed, GFP is the brightest green fluorescent reagent for this kind of assay. As I was interested in the earliest timepoint of apoptotic induction, I chose the GFP label over FITC. In terms of sensitivity the Clontech annexin V-EGFP technology may be the most sensitive on the market!
In conclusion, for investigators working in the field of apoptosis I can thoroughly recommend this product. The bottom line for many investigators is how long will it take to produce the necessary figure for that paper you're writing. Try Clontechs Apoalert Annexin V-EGFP for the detection of early apoptosis, and you'll find it won't take long.
Jeffrey Armstrong, Ph.D.
Post-Doctoral Fellow
Stanford University