I'm trying to characterize dexamethasone-modulated MPLA-stimulated murine dendritic cells for a model of arthritis and so far I've obtained a tolerogenic profile according to surface molecules (CD86, CD40). I'm having a hard time trying to detect relevant cytokines in supernatants, mainly IL-10 and IL-12p70. I have read in other publications that CD40 ligation is needed in order to detect these cytokines, and in many of them they use the J558L CD40L-transfected murine cell line. The problem is I don't seem to find it anywhere and no one I know uses it. I also noticed there's a soluble CD40L available for purchase, so I wanted to know what approach could be the better one in order to characterize the cytokine secretion profile of my murine DCs. I'd appreciate any kind of experience or help.
Instead of CD40L, you can use agonistic anti-CD40 antibody (for example,MR-1), which is commercially available from from Bio X Cell, Inc., West Lebanon, NH, USA.
For the induction of IL-12p70 it is the best to include certain cytokines, namely IFN-gamma, GM-CSF and IL-4 (for reference see PMID: 10993913, PMID: 11313382, PMID: 20975040). Addition of CD40L will also show synergistic effects if your DCs highly express CD40, but the addition of the cytokines will add strong IL-12p70 capacity. There is a commercial supply of recCD40L which you find cited here (PMID: 11313382). The MR-1 antibody mentioned above is an antibody against CD40L but not against CD40, but for stimulating DCs you want to activate CD40. You have to bind and crosslink CD40 on the surface of your DCs (not CD40L). Antibodies to CD40 (e.g. FGK4.5) bind to CD40 but are poor cross-linkers, thus you get only very poor activating signals. The CD40L must be multimeric (at least trimeric) or immobilized e.g. in the membrane of a transfected or transgenic cell expressing CD40L.
Could you also use better IL-12p70 inducing stimuli e.g. CpG-ODNs (CpG-1668 or 1826 etc)?. Murine DCs respond very well to those TLR9 ligands and they are very cheap too (in contrast to the commercially available CD40L). For ideal stimulations of murine DCs for the production of IL-12p70 have a look at the publication I mentioned above.
Sorry that I made a big mistake: I supposed to recommend anti-CD40, not CD40L, antibody.
Thank you all for your feedback. I didn't want to add anymore cytokines to the culture because that might alter the tolerogenic profile I'm looking to achieve in my dendritic cells. I found out that a human CD40L-transfected 3T3 fibroblast cell line actually works and can stimulate my murine DCs; I was finally able to detect IL-12p70 in LPS-matured DCs and also levels of IL-10 which are very low in unstimulated tolerogenic DCs. I've read there's an 78% of homology between human and murine CD40L so I will probably be able to use this cell line for future experiments.
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