I am having Propidium iodide, Acridine orange, ethidium bromide, bis benzimide. Which can be best out of this? I have read various papers which made me more confused about this. Can anybody help or suggest me the best way to do this analysis?
Using PI + annexin V-FITC is the most reliable technique. It gives you values of early apoptosis, late apoptosis/necrosis and strictly necrosis. I also suggest you to use the reagents from BD.
Hi, I have a friend who works on apoptosis. I believe he uses Annexin V for his work.
Maybe you could read about that too. Thanks.
Annexine V is the best stain for analysis of apoptosis.
However, PI stain works better, compared to other stains for analysis of apoptosis from the cell cycle graph. What is the make of the flow cyto you are using.
Using PI + annexin V-FITC is the most reliable technique. It gives you values of early apoptosis, late apoptosis/necrosis and strictly necrosis. I also suggest you to use the reagents from BD.
Dear Chudasama,
I Think that you have to make table, if you have to compaire it, it is my thinking.
I tink Using PI + annexin V-FITC in cominatiom with tunel assy. Also MMP with JC-1 gives results for apoptosis.
I would use 7-aminoactinomycin D (7-AAD) instead of PI as it will give you an extra channel to work with on most flow cytometers. PI will often be detected in the FL2 and FL3 channel, while 7AAD will only be picked up in FL3.
Hi Piyush,
Better use Propidium Iodide or PI+ Annexin FITC staining.. to see the early, late apoptosis population.
Hi Piyush,
As suggested above Annexin-V staining of phosphatidylserine (exposed on the cell surface during early apoptosis) is normally combined with a dead cell dye such as PI or 7-AAD that stains the DNA of cells with permeable membranes i.e. apototic/necrotic. As the others suggest PI and 7-AAD are common options, both activated by a 488nm blue laser. Depending on your cytometer and other flourochomes you want to use other options are To-Pro3 on the red laser and the live/dead fixable dye range from invitrogen (range of colors are available).
Hi Piyush,
Check out the following thread
https://www.researchgate.net/post/What_is_the_general_consensus_on_flow_cytometry_about_cells_with_FITC-annexin_PI
-Gaurav.
In past few years our group has modified PI based flowcytometry apoptosis assay (Nicoletti Method). You can find the detailed method in our 2007, 2008, and 2012 publications.
wich pathway of apoptosis would you like to mesure?
caspase dependent pathway?
depolarization of the inner mitochondrial membrane?
annexin V / IP is find to caspase dependent way
DiOC6 is good for the depolarisation of the inner membrane
PI and AnnexinV-FITC staining.
AnnV-PI-, live cells
AnnV+PI- early apoptotic cells
AnnV+PI+ late apoptotic cells
AnV-PI+ cells might be necrotic cells.
You can also enumerate the cells in each population using CountBright beads.
Annexin V-FITC/7AAD staining
Ann(-)/7AAD(+) dead cells
Ann(-)/7AAD(-) live cells
Ann(+)/7AAD(-) early apoptosis
Ann(+)/7AAD(+) late apoptosis
Annexin V -Alexa Fluor-488 along with Propidium Iodide will work best.
Also you could use Chicken Erythrocyte Nuclei Singlets (CEN) as an internal control.
Unlike viable cells during apoptosis, phosphatidylserine (PS) is translocated from the inner cytoplasmic membrane to the outer surface of the membrane.
I use Annexin V together with 7-AAD since I almost always need to stain for a few more markers simultaneously, and I definetely will want to use PE for those.
You may also want to check out the new amine binding fixable viability dyes instead of PI or 7-AAD. BD Biosciences, eBiosciences, Biolegend and Life Technologies sell them in may "colours". You can choose to fix your cells or not.
eBiosciences have a protocol with wich you can actually stain for Annexin-V AND fix your cells here: http://www.ebioscience.com/resources/best-protocols/flow-cytometry-protocols.htm
Annexin V for ealy and late changes in the translocation of phosphatidyl setrine and PI for late apoptosis and cell death.
Annexin V it the best for detecting early event of Apoptosis and BD sale the kit for it to rule out the necrosis.
You can use annexin V and propidium I for differentiation of early and late apoptosis, in addition the ratio of cell death can be measured simultaneusly based on propidium J staining., Reference: Meltem Demirel Kars and coworkers published years ago in the Int.ernational J.Anticancer Research. The method described in details in a number of papers.
PI to differentiate between live and dead cell ; Annexin V for apoptosis marker.
You are right Babu Mia, when you use the fluorescein labelled annexin V for early apoptosis and propidium iodine for counting the dead cells
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