ELISA IL12 p70 from mouse dendritic cells culture SN - (Aug/24/2006 )

hi,

has anyone troubles to detect IL12 p70 from culture supernatants (mouse dendritic cells) by ELISA?
i have tried some combinations (IL12 p40 capture, p35 dection; p70 capture and detection... and even a commercial kit) and never obtained significant signal in LPS-stimulated cells, but standard recombinant IL12 p70 ok!!!

any comment?

Seb

Dont panic!
It happened to me also, I got tons of p40 and nothing (or almost nothing) p70.
The point is: IL-12 secretion is mediated by T-cell interaction. What i mean is, you will not detect IL-12p70 unless you add T-cells or anti-CD40, gotta?!
Good Luck!
Diogo

PS: wich DCs are you working with?!
Splenic or BM ?
I need some help with rGM-CSF concentrations for BMDCs, can you assit me?

thank you a lot Magnani,

so, don't you think that DC produce IL23 since p40 is shared by the 2 cytokines?
I have already observed that LPS, as well as CD40L and a-CD40 (FGK), upregulated IL12 p40 mRNA
i will check IL12 p35 and IL23 p19 next week...

i'm working with BMDC and the D1 cell line
i usually use rmGM-CSF from USBiological; it works well at 2 ng/mL in 5% FCS

Seb