I wanna research whether some compounds effect on the inflammasome, especially NLRP3. Considering the raw264.7 donot expreesion ASC, can I use the ASC plasmids-transfected cells (not stable expression) ? Besides, can I use the BV2 cells ? Did anybody ever try these methods ?
One can restore the expression of ASC protein to RAW264.7 cells. However, one can use J774 cells (mouse) or human THP-1 cells. Further, many epithelial cell lines also can be used to study NLRP3 inflammasome activation after "priming".
Thanks, Divaker. Since I have no J774 or THP-1 cells, so I wanna transfect the ASC to Raw264.7, but the stable expression needs more time and energy, can I just activate the NLRP3 at 24-48h after transfection.
The ability to detect mature IL-1beta and/or IL-18 in transiently transfected cells with ASC cDNA may be limited. This is because only a tiny fraction of cells are transfected. Therefore, it may be very challenging to get reproducible results in the transient transfection coupled inflammasome activation assays.
The problem lies in the fact that transfection of macrophages usually leads to some level of toxicity. The cells that will have ASC expressed in them will suddenly have a functional inflammasome pathway and will be more susceptible to pyroptosis via activating stimuli released by neighboring cells dying from the transfection itself. It's a sort of "catch-22." However, it is possible. I have successfully stabilized RAW264.7 with ASC-EGFP in the past, but as Divaker said, it is very low transfection to begin with. Maybe 3-5% with ASC (versus 50-70% with just EGFP). After stabilization or flow sorting, however, you can get quite good numbers and the cells can be used to study NLRP3 signaling.
BV2 also do not express ASC. So BV2 cells do not activate ASC-dependent inflammasome, such as NLRP3, AIM2 et al.
Sorry, Do-wan. Actually, I have tried the BV2 cells, and I did find the IL-1beta in the cell supernant. I think BV2 express ASC. Besides, I find a literature in Pubmed "Inhibition of phagocytosis and lysosomal acidification suppresses neurotoxic prion peptide-induced NALP3 inflammasome activation in BV2 microglia".
Thank you, Divaker and Jordan. As you said, it is not very easy to get results in the transiently transfected cells, I decide to use the BMDM primary culture. Thanks for your advice.
- Badges
- Science topic