Dear Lindsey St. mary,

We used to prime human PBMC/THP 1 cell with LPS (1 ng/ml) for 3 hours followed by ATP(5 mM) for optimum IL-1beta release.

Regards,

Manoranjan

The best answer is "probably" yes. Your readout will be the deciding factor. If you are looking for:

• Specks by microscopy
• IL-18 release by ELISA/WB
• Caspase-1 cleavage by WB
• ASC dimerization by WB

Then yes, it would probably work. The following paper showed that licensing of the inflammasome is rapid and only requires on the order of 5 minutes to engage:

http://www.jimmunol.org/content/192/8/3881.full.pdf

Effectively, the inflammasome requires post-translational licensing. The long duration priming that most people use is usually to synthesize IL-1b and additional amounts of components such as NLRP3. However, NLRP3 is present in most cells at a low level constitutively and IL-18 is definitely constitutive. As such, a brief priming with LPS or some other "signal 1" stimulus will be sufficient for NLRP3 inflammasome priming.

Using them together would probably have the intended effect, but you run the risk of the P2X7 engagement with ATP throwing the system off of balance since it is detected and responded to within a few 10s of milliseconds. It's best to do a one-two punch with a brief 5-minute priming phase followed by ATP.

I agree with Jordan's comments and would like to add that NLRP3 activation works differently in myeloid versus non-myeloid cells. In your case, when you use LPS (100 ng/ml) and ATP (5 mM) together for macrophages, it would be desirable to have a time course to follow activation of caspase-1 (generaton of p20 and p10) and secretion of IL-1beta (p17).

Yes, you may use two stimuli at once. In this case your readout should be Il-18 and it should not be much different from two subsequent hits. We tested this in our lab. Just my 2 cents to the previous very detailed answers.

However, as Jordan and Divaker suggested, two separated hits (first LPS and then ATP) is preferable.

I have a question about priming.

As Manoranjan stated "LPS (1 ng/ml) for 3 hours followed by ATP(5 mM) for optimum IL-1beta release". So do you change the media to remove LPS, when you add ATP or you keep the media containing LPS?

Changing the medium is the easiest way to do it. Remember that the first signal (or hit) is the induction of IL-1b and IL-18 mRNA transcription and translation of pro-IL-1b and pro-IL-18, which is a regulation step that takes longer to take place (that's why you need at least 3 hours interaction with LPS). The second signal results in Caspase 1 activation and cleavage (therefore activation) of pro-IL-1b and pro-IL-18 into their active forms. This process being a much faster regulatory process.

My point being that the first signal generated by LPS takes much longer than the second one (generated by ATP). So any additional effect of leaving LPS while you have the second signal would be most likely negligible.

I hope it helps. Cheers.

Hi Daniel,

Thanks for your reply. I am trying a time course analysis and if I keep LPS stay for 7hrs, will it not affect my results in untreated controls?

Il-18 is constitutively expressed, as well as major inflammasomes partners. To prime monocytes, you don't need mRNA and new protein synthesis. LPS priming for 5 minutes  followed by ATP   is sufficient to prime and stimulate inflammasome resulting in cleaved il-18, p20 caspase-1 and many other inflammasome proteins release.

Indeed, IL-18 and IL-1beta have different expression patterns. But if you want to analyze IL-1b you have to use longer times priming with LPS.

You can find some really nice protocols in the chapter 4 of the publication below.

Methods Mol Biol. 2013;1040:29-39. doi: 10.1007/978-1-62703-523-1_4.

Assessment of inflammasome activation in primary human immune cells.

Plantinga TS, Joosten LA, Netea MG.

http://www.ncbi.nlm.nih.gov/pubmed/23852595

Your untreated controls shouldn't show any differences in the release of the mature form of these cytokines.

I hope it helps. Cheers.

http://www.ncbi.nlm.nih.gov/pubmed/22948162

see figure 1, especially panel A. :10'LPS followed by ATP

then see figure 1 panel B.   :ATP then LPS, or simultaneous LPS + ATP lanes...

It should explain your queries.

http://www.ncbi.nlm.nih.gov/pubmed/22948162

cab anyone suggest me which LPS will be effective for THP1 priming for IL-1B priming and NLRP3 activation .E.Coli or S.aureus or anyother starin of bacteria.

Kindly suggest me.

Thanks in advance .