Recently, I use CpG oligonucleotide for IL-12 ELISA.
I made CpG RCA(Rolling Cycle Amplification) product and treat 5ug/ml to Bone Marrow Dendritic Cell.
After 48h later, I did ELISA by using supernatant, but result was almost zero..
IL-12p70? Or are you looking at one of the subunits? Try looking around 18 hours or so for p70. And look for p40 at later time points. You can also look at gene expressions of IL-12 subunits to see if you see any induction.
Let's do the obvious stuff first... is your cell concentratoin around 1x10e6 cells/mL?
Did you try a different CpG?
And also, CpG isn't a great stimulator of IL-12 or much of anything else by itself.
Hi,
I would look at the IL-12 production by DCs much earlier than 48 hours. I think it should peak between 8 hours and 24 hours.
Best,
Vural
Thanks for all of your answers.
I used Bone marrow derived DC from BALB/c mouse.
I tried to detect IL-12p70 by using CpG RCA product, but failed. And also I used just CpG oligonucleotide, too. But LPS, which is used for positive control, had somehow showed some result.
And does the time effect the result? As I know, ELISA is just detect the amount of cytokine (or somewhat you want to detect) in solution, so the released cytokine would remain in solution..
The released cytokines will not just accumulate in the supernatant. Those are live cells you are using, and they continuously take up proteins in the supernatant. That leads to secondary and/or enhances activation and further cytokine/chemokine release.
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