Hi,
is it possible to activate inflammasome in mice BMDM cells wich are not primed with LPS?
We are trying to search for increase of Il1ß after cholesterol crystals incubation but we do not see any effect.
Maybe I do not undestand properly, but there are two ways to activate NLRP3:
1) classical two step activation which requires requires a priming step that can be provided by i.e. LPS, and which activates NFkB-dependent pathway of transcription, and second signal (i.e. DAMP - like cholesterol crystal)
2) non-transcriptional priming: rapid way, which can be caused by DAMPs
In this situation, is it enough to treat the macrophages only with cholesterol crystals which will act as a non-transcriptional primer and than as an activator? Or am I missing something?
Best!
Hello, as I know cholesterol crystals can be used as activator (signal2) after a step of priming with LPS (signal1). Since I did not see yet any paper showing that cholesterol crytals can both prime and activate the NLRP3 inflammasomes, so my suggestion would be, first try to prime mouse BMDM with LPS and use cholesterol crystals as activator to confirm that your cholesterol crystals are working fine and have an effect on the cells you are using. Because sometimes the problem can reside in the experimental conditions: cells, treatement... Second if everything works fine, so maybe you can change the concentration of cholesteerol crystals with which you treat to increase the activation (checking other papers can help you with the dose and time you might use for your experiment). Finally as I mentioned it is the first time for me to hear about such role of cholesterol crystals so maybe it will be a new finding if you succeed in proving it, then maybe you have to try to make your own experimental model or setting to demonstrate this interesting hypothesis..
Good luck
Dear Iskander Madhi
I have one more question - do you remove the media after 3 hours priming (and before ?
Best regards,
Michal
Yes I change media before priming, to a media without FBS because if you keep the FBS, you will have a huge pellet after the precipitation of your proteins which is not good to get good detections for your IL-1beta or caspase-1 in the supernatant.
Also, priming time differes depending on the concentration of LPS you will use, some papers did only 30 minutes for priming. So, maybe you can optimize time and concentration of LPS to your model. I usually do 1ug/ml LPS for 3 hours.
Good luck!