I would like to measure the activity of FAS/TNFR-dependent pathways and apoptosome-dependent pathways separately using FACS or a fluorometer. I do NOT want to use Western blot.
The best solution that suits me is measurement of:
- cleavage products of activated caspase 8 (for extrinsic) and
- cleavage products of activated caspase 9 (for intrinsic).
There are some good antibodies that work well with FACS and would allow me to quantify the activity of both pathways. However, I would like to ask, is this a correct approach? Have you tried quantifying cleavage products and did you get comparable and reliable results?
There is an old method for intrinsic pathways that I don't really like. It is common to extract cytoplasmic and mitochondrial contents in separation and detect cytochrome c with an antibody. I wouldn't like to use this method and would prefer something that could be strictly related to caspase activity.
And yes, I have read about FLICA-FAM compounds, but they are a bit controversial according to some articles. I don't want to base my results on them until somebody disproves their unreliability.
You could use an antibody to cell death receptors, ie FAS. to examine intrinsic pathways.
just because the FAS receptor is there that doesn't necessarily mean that it is being enganged and activated. My suggestions was going to be a commercial ELISA colorimetric assay for caspase 8 and 9. I think this approach would be valid. Cell Death and Differentiation Journal's latest review would agree with it at least.
Can you share any experience with using ELISA or luminescence-based detection of caspase activity? This is a very good idea.
Yong Weon Yi, I have had those kits on my mind for some time now. I think it's a good solution. I was a bit conservative because in some suspicious journals there are articles critisizing those short peptide sequences as highly unspecific and then the information was copied over and over in the next articles about caspase detection. I am sure we can trust Promega with their kits and quality assurance. Thanks for the answers.
You should be aware that caspase 9 can also be activated by extrinsic pathway via caspase 8 mediated bid processing. To really discard the involvement of extrinsic pathway you should block specifically casp 8 or disc formation and test if cells are still dying. The best alternatives would be to overexpress the viral serpin crmA that is very specific for casp8.
OK. What reagent do you recommend for blocking caspase 8 and caspase 9?
What about the colorimetric kits from Biovision?
Are they reliable?
Did anybody have used it?
I've read articles from Nature using both Biovision as well as Promega's luminescence, with the majority using the latter one. Plus it's quite easy and give good, replicable results. Actually, many people still use Western blots which are the least reliable in my opinion.
this is right, caspase 9 can also be activated by extrinsic pathway via caspase 8 mediated bid processing. But you can study other important proteins of extrinsic and intrinsic pathways along with the caspase family.
Dear Filip,
Greetings! Its is good to detect caspase-8 for extrinsic and caspase-9 for intrinsic pathway of apoptosis. But you should check that the extrinsic pathway will be depended on RIP3 or not. If yes then caspase-8 and RIP3 (FASL mediated) both are important to measure the extrinsic pathway of apoptosis. But if RIP3 is recruited through TNF then u should check for NFkB to confirm apoptosis.
About intrinsic pathway with caspase-9, tBID is good for the detection of intrinsic apoptosis. Though CtyC is a good factor but as you would like to escape that way, u can choice tBID. without FACS u can do immunoprecipitation to prove the pathway.
like IP with cas-9 and IB with Cas-3 and IP with cas-8 and IB with cas-3 to support your FACs data.
Wish you the best,
I would not be critical, but there are too many interactions between the intrinsic and extrinsic pathways in apoptosis. Therefore, measure ONLY one pathway, whatever method you want to use, it is virtually impossible and your results, IMHO, will lack credibility.
If you want to inhibit caspase-8 you can overexpress crmA which is an inhibitor of caspase-8. To measure intrinsic apoptosis you can measure the activity of Bax and/or Bak by conformation specific antibody (e.g. clone6a7) using FACS. These proteins might also be activated if you add the FAS ligand because tBiD is probably also generated. often this protein is hard to detect because of the available antibodies and endogenous expression levels of bid. In addition tBid is not generated in all the cell types (see TypeI/TypeII cells). If you see activation of Bax or Bak (make kinetic) by FAS you can overexpress e.g. Bclxl to block the intrinsic pathway. If the cells still die by FAS this you can quantify by staining for active caspase-3 (FACS). As a general positive control to show that the intrinsic pathway is active/ inactive use e.g. Staurosporine, or overexpress the BH3 protein Bim.
Dear Filip
As already some of our interactors said, it is quite difficult to measure the pathways independently as in majority of cases, they are linked or connected. However, this is almost the question we have asked in our last year paper "Thomas et al. (2013): Upregulation of DR5 Receptor by the Diaminothiazole DAT1 [4-amino-5-benzoyl-2-(4-methoxy phenyl amino) thiazole] Triggers an Independent Extrinsic Pathway of Apoptosis in Colon Cancer Cells with Compromised Pro and Antiapoptotic Proteins: Apoptosis, 18, 713-726". This might be of help to you. Best of lack.
Another thing is, I don't think you can avoid Western blots.
An simple approach can be found at:
Assessing main death pathways in T lymphocytes from HIV infected individuals.
Massanella M, Curriu M, Carrillo J, Gómez E, Puig J, Navarro J, Dalmau J, Martínez-Picado J, Crespo M, Cabrera C, Negredo E, Clotet B, Blanco J.
Cytometry A. 2013 Jul;83(7):648-58. doi: 10.1002/cyto.a.22299. Epub 2013 May 6.
PMID:
23650261
[PubMed - indexed for MEDLINE]
I agree with the others, due to the interactions between the 2 pathways it's nog enough to only look at Caspase 8 and 9. You also need to take a look at some other markers within the pathways. You could also use the caspase inhibitors zVAD/LEHD/LETD/etc to show the role of the caspases/pathways. An other option could be the RT-MLPA R011 apoptosis kit from MRC Holland. This can give you a general overview of the apoptosis markers that play a role in you're samples and from there you can choose the rest of you're exp.
Specific caspase substrates from Calbiochem can also be used to check the activity of caspase 8 and -9.
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