I stimulate THP-1 macrophage with LPS (10ng/ml-1000ng/ml) for 24h, but detect no IL-1beta and IL-12 in supernatant.
Someone said LPS just increase pro-IL-1beta expression inside cells since secretion of IL-1beta need clevage of pro-IL-1beta. So I need add something like ATP with LPS to cleave pro-IL-1beta.
But why no IL-12 can be found in supernatant?
We do it on a regular basis. We can detect 4 ng/ml of IL-1beta 18 hours post stimulation of 2 million THP-1/ml with 1 ug/ml of LPS ( without any ATP). Cells should be healthy, growing in a log phase ( no older than 2 months in a culture). Cells should not be mycoplasma contaminated as mycoplasma makes cells intolerant.
you may add 2-5 mM ATP for the last 30 minutes to trigger NLRP3 inflammasome. In this case 3 hours of LPS will be sufficient.
Strange. Yes we can also detect IL-1 beta in THP-1 cells which are stimulated with LPS. There must be something wrong in your exptt. like contaminations etc..
Have you differentiated the cells with PMA? Undifferentiated cells express low levels of IL-1b mRNA but is markedly upregulated with PMA treatment.
Thank Grant. I had added PMA to differentiate THP1 for 48h before LPS stimulation.
Mikhail. Thank you for your detailed answer. I plan to detect mycoplasma to exclude contamination. Do you mind send me your protocol?
Thank Manisha. I plan to detect mycoplasma. Could you send me your protocol?
Shujun,
Please see my publication in PLOS ONE, 2010 ( Zakharova et al). There is detailed protocol with all primers etc. if you have more questions, I will be happy to answer.
good luck!
Dear Shujun,
can you please provide some more information on your culture conditions? In particular on concentration of PMA and time of maturation. It has been shown that high dosis of PMA activate THP-1 cells so that their resposne to LPS is attenuated.
Best Stefan
Agree with Stefan. If you use 100 nM and more of PMA ( I didn't test for lower doses), you will see induction of IL1 B. Both message and protein are going away by day 5 after PMA treatment in our hands. This is the time when THP really matured to THP-derived macrophages (TDM). We use THP/TDM as a quick surrogate of Mo/MDM and characterize them quite well.
Dear Mikhai,
I stimulated THP-1 with PMA(200ng/ml=320nM) for 36h. After 24h incubation with LPS(10-1000ng/ml), we detected IL-1beta and IL-12 and found no increase compare with negative controsl. Mycoplasma contamination maybe a very important problem since we cannot find it directly. I plan to test TNF-alpha and mycoplasma. According to your paper(figure 3), TNF-alpha expression induced by LPS wouldn't decrease with mycoplasma contamination. Is it right?
Dear Stefan,
I stimulated THP-1 with PMA(200ng/ml=320nM) for 36h. After 24h incubation with LPS(10-1000ng/ml), we detected IL-1beta and IL-12 and found no increase compare with negative controsl.
If your THP-1 cells are PMA-differentiated then there should only be cytosolic pro-IL1b, as macrophages do not readily release mature IL1b and pro-IL1b is tightly regulated and maintained intracellularly. In this case you will need a second stimulation, the most rapid being extracellular ATP (1-5 mM) or nigericin (5-20 µM).
Non-PMA-treated THP-1 cells (monocyte-like) should release mature IL-1b with LPS treatment alone.
I cannot comment on Jordan's suggestion as I have never tried this setting. From my point of view, 200 ng/ml PMA is too much. Standard protocols suggest 100 ng/ml but this already causes activation. Try between 10 ng/ml and 20 ng/ml; unfortunatey, the concentration dependes on your culture conditions (e.g. cell density), batch and producer of PMA, etc.
Shujun,
Yes, up-regulation of TNF by LPS was not affected by Mycoplasma contamination as we showed in our article. However, you stimulated THP-1 with PMA and on a top of PMA stimulation you may not see extra effect of LPS stimulation. We often see similar pattern with monocytes from sepsis patients. Cells are already activated and tolerant to another stimulus. In addition, low dose of LPS in FBS may also induce tolerance. This phenomenon is well described for TNF gene. I think you should check your experimental set up step by step to find and eliminate the problem.
hey, Does anyone guide me that how to stimulate IL6-R in differentiated THP-1 cells???
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