Can I be provided with the simplest protocols for determining apoptosis versus necrosis using Flow Cytometry?

Big E said:

Can I be provided with the simplest protocols for determining apoptosis versus necrosis using Flow Cytometry?

the Purdue site
http://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

Big E said:

Can I be provided with the simplest protocols for determining apoptosis versus necrosis using Flow Cytometry?

Hi,

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-FIT