i add 4 different concentrations of LPS（0ng/ml, 10ng/ml, 100ng/ml and 1000ng/ml）into culture medium of THP-1，i used the Human Inflammatory Cytokine Kit (BD bioscience) to detect the cytokine levels but i cannot detect cytokine expression (IL-6, TNF, IL-1β, IL-10 and IL-12p70 ) within 72hr(2hr, 4hr, 8hr, 16hr, 24hr, 48hr, 72hr). Whether I need to pretreat the THP-1 by using PMA？
Can you answer some concerns? They are following:
What are the cytokines that you're measuring? Which kits are you using for measuring them? What is the source of your LPS?
You may take a look at the following article, if that's helpful for you:
http://iai.asm.org/content/68/12/6663.full
Regards,
Debaki.
what is the rational for your LPS concentrations? I would use at least 10ng/ml LPS (eg. 10x your highest concentration), I've seen experiments going up to 100ng/ml so I would probe for a range in between 10 and 100ng/ml for your next experiments.
You should added the appropriate volume to reach a final concentration of 1 ug/mL. You can see this article
Article In vitro immunomodulatory activity and in vivo anti-inflamma...
You are currently using LPS at very low concentration. There are several latest research articles that you can refer for working concentration of LPS. Also, look at the baseline cytokine detection levels of your cytokine kit. You might have to further increase LPS concentration to observe target cytokine release using your cytokine kit.
As you know, THP1 cells are monocytic. If you are interested in finding the cytokine levels in macrophages, first induce your THP-1 cells with PMA. Again, you can take reference for PMA concentrations from recent research articles.
These references might be helpful :
https://search.proquest.com/openview/1fc911b7814803ef0629a21a2539b3c1/1?pq-origsite=gscholar&cbl=40569
http://www.sciencedirect.com/science/article/pii/S1097276502005993
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3793604/
THP-1 cells do not express CD14 constitutively. Thus, without pretreatment with PMA, it is fully normal to not get any cytokines expression.
Pretreat your cells with PMA 100-500nM for 3h before activation with 1ug/ml of LPS. You will see all cytokines expression that you want!
Good luck
Greetings Ya! Keep in mind that THP-1 are promonocytic, which means that they can be induced to differentiate or "mature" into monocytes. However, because they are promonocytic, they do not express CD14, the receptor for LPS, therefore, currently, you are treating these cells with LPS and they have no way receiving it into the cell. While CD14 is implicated in the induction of the expression of a number of cytokines, you should have gotten some detection of at least IL-1B, IL-6, and TNF, at least, even without priming the THP-1s to maturity. My suggestion, pretreat/prime your cells with PMA (I used 50ng/mL in my research), this will get you the induction of CD14, so then you can treat with LPS. Then try measuring your cytokine expression again.
Hope this helps and best wishes in your research!
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