I primarily see ASC oligomerization assays (with DSS crosslinking) done in cell culture collections, but I have been unable to find any examples of this assay being done in tissue homogenates. Is this possible? Does anyone have a suggested protocol?
Hope this helps!!!http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3494892/
Thanks for the reference, Sandhya. That paper has some useful methods in it, actually, but they do not do an ASC oligomerization assay. The assay results look as follows (labelled ASC monomer, dimer, trimer, etc):
http://www.nature.com/ni/journal/v11/n5/images_article/ni.1859-F3.jpg
Greeting, Jordan.
to see ASC oligomerization, inflammasome (mainly NLRP3) should be primed and activated with signal 1 and 2, respectively. Do you see monomeric ASC, NLRP3, TLRs, P2X7, ERK in tissue homogenates?
Hi Mikhail,
Yes usually I would do signal 1 and signal 2 to stimulate ASC oligomerization, but the current system isn't amenable to that treatment because these are primary in vivo tissues. Consider hypothetical tissues that are coming from a system with elevated serum IL-1beta. I am looking to see if those tissues have dimerized/trimerized ASC. I have not yet tested these tissues for monomeric ASC, etc, but it is there because macrophages are confirmed in the tissues.
I will have some precious sample soon, a portion of which I am considering crosslinking with DSS to look for dimerized/trimerized ASC. I was hoping that maybe someone would have a protocol for performing this assay on tissues, but I have not yet found one. It is almost always on purified macs/DCs.
I think you will be the first one. We also work with monocytes and macrophages. However, we could see released ASC in human lung BAL but never checked for oligomerization.
Good luck!
I am also working with ASC and I have tried the last period to take oligomerized ASCs after DSS treatment in tissue samples. I figured out a protocol on my own and at first I just took the inflamed region of the tissue and after protein isolation with special buffer and crosslinking, I resulted in the oligomerized forms. When I did it in the whole tissue, things did not go such easier. An extra issue I have is that the pellets are dissolved in SDS sample bf and I cannot do my Bradford measurement.
Jordan, what about you? Did you find anything helpful? Does anyone have an idea?
Hi Theodosia im really interested in the crosslinking protocol that you are using to crosslink ASC in tissue, what tissue are you using? Actually im doing this in primary microglia and works really good and i want to try in brain tissue
Hi Theodosia,
I haven't yet attempted this because my tissues are archival and precious. I was hoping to get my hands on a protocol that someone has tried, first. Like Eduardo, I am interested in your protocol. Would you mind posting it?
Thanks!
- Badges
- Science topic