What are the differences between using activated Caspas3, AnnexinV or Tunnel assay for evaluation the apoptotic cells? and which is better for double immunolabelling?
The tunnel assay detects apoptotic DNA cleavage. The drawback is that the tunnel assay may also detect cells having DNA damaged by other means than in the course of apoptosis.
Annexin V staining: Apoptotic cells become Annexin V positive due to phosphatidyl serin exposure. Annexin V binds to apoptotic cells while the plasmamembrane is still intact. However, necrotic cells with permeablized plasmamembrane are Annexin V positive as well. Therefore, Annexin V should be combined with PI or 7AAD staining. Annexin V positive and 7AAD negative cells are apoptotic and double positive cells are necrotic or late apioptotic.
Caspase 3 cleavage: Caspase 3 activation is a point of no return. Cells will undergo apoptosis. In rare cases cleavage is not the same as activity. Therefore caspase activity should be analyzed in addition.
Hi,
Perhaps this article will help you;
Comparison of immunohistochemistry for activated caspase-3 and cleaved cytokeratin 18 with the TUNEL method for quantification of apoptosis in histological sections of PC-3 subcutaneous xenografts
Thanks so much
The tunnel assay detects apoptotic DNA cleavage. The drawback is that the tunnel assay may also detect cells having DNA damaged by other means than in the course of apoptosis.
Annexin V staining: Apoptotic cells become Annexin V positive due to phosphatidyl serin exposure. Annexin V binds to apoptotic cells while the plasmamembrane is still intact. However, necrotic cells with permeablized plasmamembrane are Annexin V positive as well. Therefore, Annexin V should be combined with PI or 7AAD staining. Annexin V positive and 7AAD negative cells are apoptotic and double positive cells are necrotic or late apioptotic.
Caspase 3 cleavage: Caspase 3 activation is a point of no return. Cells will undergo apoptosis. In rare cases cleavage is not the same as activity. Therefore caspase activity should be analyzed in addition.
A few things to add to Karsten's answer, it really depends on what you are staining and what you want to double immunolabel.
For cleaved-caspase 3, you're performing a pretty standard immunofluorescence, so you can fairly easily add in another primary and secondary antibody. The issue with Caspase staining also is that the antibody itself is a bit tricky and often gives a lot of background, making specific quantification a bit tricky. All of this is manageable, though, if you block well and do your primary antibody incubation in a cold room overnight. It's a standard assay in my lab.
TUNEL staining is a bit more complicated of a procedure, but a lot of people consider it the gold standard, and the images are very clean. TUNEL staining will also show more dying cells than caspase staining, generally.
One other caveat regarding Annexin V is that while phosphatidyl serine exposure is usually a marker of apoptosis, it's exposed very early in the process when cells aren't necessarily committed to dying. Some people think of it more as a marker of extreme cellular distress rather than a marker of apoptosis, as phosphatidyl serine exposure is reversible in a number of contexts.
Thank you all for your answers. I have a feeling that cells positive to cleaved-caspase 3 are not stained with other antibodies in the double labeling staining. So I have a proplem to identify these dying cells. Could anyone explain why the cells are not more expressing other markers?
I will appreciate if anyone has any suggestion to overcome this proplem and tell me what is the best way to identify these dying cells.
Thank you in advance.
What marker are you staining for? I stain against cleaved caspase and other markers often enough. If you suspect that the marker is lost as the cell becomes apoptotic, then maybe an early stage indicator of apoptosis like Annexin V would work best
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