Can I be provided with the simplest protocols for determining apoptosis versus necrosis using Flow Cytometry?
Big E wrote:
the Purdue sitehttp://www.cyto.purdue.edu/flowcyt/research/cytotech/amfc/data/page2.htm
has an informative article in regards to this
METHODS IN ANALYSIS OF APOPTOSIS AND CELL NECROSIS
by Zbigniew Darzynkiewicz
So there is a dye that has been around for some time called (i believe JC-1) it is a mitochondrial dye used to measure membrane potential (or changes)--which are often early signs of cells undergoing apoptosis. A number of companies offer this kit and optimized protocols for using it in flow, as well as other applications. This might be worth looking into, in conjunction with a complementary assay for necrosis. The protocols that i have seen seem pretty straightforward (in fact I have just put together one for our company) but needs to be validated for your model system. There seems to be a large body of evidence and references regarding the use of this dye for these applications. I can provide them offline if you wish. you can message me through SS, or simply google the term JC-1.
Good luck on that one!
The best, and most commonly cited, method of determining apoptosis ina cell culture is to stain the cells with Hoechst Dye 33258 and then view them with a fluorescent microscope. If you stain the cells in conjunction with Sytox green, you can then (using different wavelengths obviously) count the number of cells that are apoptotic and necrotic.
for flow cytometry, use annexin-V-FITC.
one of the early apoptotic signals is transport of phosphatidyl-serine (PS) to the outer leaflet of the cell membrane.
Annexin-V binds PS, and so with FITC conjugated Annexin V you can use FACS to differentiate apoptotic to necrotic/live cells.
Let me add to the last post: use Annexin-V FITC with propidium idodie staining (1-2 micrograms/ml in the final suspension solution). Molecular probes sells the PI and Annexin V can be purchased from Caltag (both companies now owned by Invitrogen). Note that staining with annexin V requires a special buffer as indicated by the manufacturer. http://www.caltag.com/pdf/L12004.pdf
I generally use a dot plot showing forward scatter vs. PI staining in FL-3. Live cells are PI negative. Dead cells are brightly PI positive. The stuff in between is dying. Annexin V staining lets you distinguish those that are dying from apoptosis from those that are dying from necrosis. If you want to convince yourself, you can always expose some cells to hypotonic solution to stimulate necrosis.
I also plan to use annexin V and PI to stain apoptotic cells. But I don't see how you can use it to disinguish between apoptotic and necrotic cells. Woun't the cell membrane rupture and PS be accessible for annexin-V staining during necrosis also?
In my head, it can only be used to distinguish between dead and living cells...
Add a fluorescent DNA staining agent like propidium iodide. No DNA is exposed during apoptosis so the apoptotic cells won't be stained. But the necrotic cells will.
Have you tried the APOPercentage apoptosis assay. It's a quantitative dye based assay. Transfer of phosphatidylserine to the outside of the membrane permits the transport of the dye into the cell but necrotic cells cannot retain the dye. http://www.biocolor.co.uk/index.php/assay-kits/apoptosis-assay-1/
The APOPercentage assay has been adapted for use with flow cytometry. BioTechniques 45: 317 - 320 (September 2008)