Does anyone have a protocol for stimulation of THP-1 cell line with synthetic CpG-ODN that stimulates this cell through TLR9 signaling pathway? I was trying without differentiation with PMA and with prestimulation with different concentration of PMA. Without prestimulation they do not respond, but in case of PMA treatment I get IL-8 and TNFa response even in nonstimulated conditions.
Indeed, I have enough experience with THP-1 cell line but from virological point of view, I do not know too much about CpG-ODN,
but im my case, sometimes the THP-1 cells stimulated in case of bacterial infection, this stimulates the cells,
check your media, handling condition.
Hopefully, this could help.
In some cases (dont exactly know why..yet)..to see the effects of TLR9 stimulus in THP-1 you need to have an additional trigger (i.e.LPS). If you repeat your experiements with ODN, LPS and ODN+LPS you might see that ODN stimulus enhances your LPS effects (even synergetic). Additionally, its important to check several different types of ODN (Type A,B,C etc)..if you use bacterial DNA it might work better just because that is inherently contaminated with other MAMPs from the source bacteria (so there you already have your additional other TLR trigger).
On another note, if you use PMA differentiation, it takes a long time (depending on how much PMA was used) for the cells to recover as in not produce cytokine anymore. (see attached article)
Hope this was helpfull.
Hi Juliet! Yes, I managed to optimize my protocol. So in my hand the best way was to pre stimulate cells with PMA (10ng/ml medium) over night (approx 16 h), wash them once with medium and stimulate with CpG2216 for 5h! 2006 was not working in my hands (signal was not as high as with 2216) and best read-out was TNFa expression in supernatants (ELISA, eBioscience)! Cells were cultured in 24 well plates, 1x10 5 per well and stimulated with 10 uM CpG2216! Hope it will help you! Take care with passage number, in higher passages they were totally strange - no stimulation!
Wish you to have good luck with your experiments ;-)
Thank you so much for the details. Do you know if IL-10 is produced using your strategy?
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