If you want to analyze apoptosis and necrosis cells at ones try to employ Vybrant Apoptosis Assay Kit. In Q2 frame you will get both population of cells you want to study
When you stain cells for FACS you can use Annexin-V and 7-amino actinomycin D (or propidium iodine). A cell that stains positive for Annexin-V but is negative for 7AAD would be early apoptotic. A cell that stains for both Annexin-V and 7AAD could be apoptotic or necrotic.
Hello Lei,
The most conclusive way to identify a necroptotic cell is to utilize necrostatin to block the cell death. While you can use annexin v and a cell death permeability stain such as 7-AAD or propidium iodide, differences between necroptotic and apoptotic cells are not clearly defined using this method.
If you are doing in vitro work, I would suggest adding necrostatin (which blocks RIP1 kinase) to your culture and see if it is able to abrogate the death of your cells. If you are doing in vivo work, I would suggest sorting your cells and examining PARP cleavage via western blot. A differerent signature of PARP cleavage has been identified to correspond with necrotic rather than apoptotic cell death. As for flow cytometry, I would say there is no clear method of identifying apoptotic vs necroptotic cells at this point, due to significant overlap in the signatures of each.
One thing I have used that might indicate necroptosis via flow cytometry, is to dual stain for DCHF-DA and Propidium
Iodide. An elevated DCHF fluorescence would indicate elevated reactive oxygen, which is associated with necroptosis. This is definitely not a conclusive technique though.
yeah, I also think about that method.But, necroptosis is not a simple.It looks like a necrosis,but it programed by some cytokine, for instance, RIP family.So, I really want to study these kind of program necrosis cell under different impairment. I found some paper to focus on necroptosis,but how to identify these cells on slides is difficult to made.the attachment is one paper to identify by western blot.
How to made it positive on slides??so difficult.
Ahhhh, ok. I have had some success looking at immunofluorescent staining of RIP kinase and looking at cell localization. Early in necroptosis Ive seen an increase in localization at the cell surface (both RIP1 and RIP3). Also you could do a TUNEL stain, where you don't usually get the nice bright condensed nucleus that you would see in an apoptotic cell. Rather necroptotic cells have an atypical TUNEL staining, but you would need to a good apoptotic control to compare with.
You might try the methods I outlined above, but in the end I think you'll need to supplement with western blot to confirm. With western you should be able to see increased expression of both RIP1 and RIP3. Also, you can sometimes see a second slightly slower migrating band for RIP1 that corresponds with phosphorylation of RIP1.
I agree with Scott that there is no clear method of identifying apoptotic vs necroptotic or necrotic cells. Propidium iodide is a standard dye which translocates into necrotic or late apoptotic cells. It seems to RIP kinase reflect special potential to study necroptosis, but of course for publication you will have to compare your results with apoptotic control
i thik if only is a cualitative semi cuntitative test use anexine V but if you like more especific responses use flow cytometry with PI or FITC that is more cuantitative but need standarization depens of the cellular line
The criteria for using FACS staining to differentiate early apoptotic from late apoptotic/necrotic cell is the integrity of cell membrane. In addition to using annexin and 7AAD/PI staining, you can also use higher dose of 7AAD (20 ug/ml vs. 1 ug/ml) to identify early apoptotic and the later apoptotic/necrotic cells. There were some papers looking at apoptosis of lymphocytes in HIV+ people using this later method
RIP3 expression is elevated during necroptosis could be seen as a direct marker. See figure 8 in 23348743 [PMID].
PARP/Caspase cleavage Vs elevated RIP3 expression would give a clear cut answer.
http://www.ncbi.nlm.nih.gov/pubmed/23348743
RIPK1, RIPK3 and the more direct target MLKL(total, and Phospho)protein level would be good assay, concomitant with Caspase 3 cleavage product increase. If examining in vivo,tissue calcification by von kossa staining would give more clear scenario.
Hello!
Is it true or false, if we say the morphological characteristics of necroptosis are same with the morphological characteristics of necrosis? So that, the flowcytometry apoptosis annexin-v will record necroptosis as necrosis cells.
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