I am looking for the best and most practical way to stimulate/activate DCs via CD40L stimulation. The most practical seems soluble recombinant human CD40L (2 ug/ml) - however, it does not seem to do the trick.
We need it for subsequent IL-12p70 detection and with soluble rhCD40L - the responses are very poor?
My experience tell me cell contact CD40L is better than soluble ie CD40L in the fibroblast stimulate better and also depends on many factors for soluble CD40L to work well such as how you culture DC,donor difference, quality of CD40L (different company products) and dose response to different donors since DCs are sensitive cells. Any small change would make difference.
CD40L is effective in 'switching' antigen-presentation from class II to class I for stimulation of CD8 CTLs. However, by itself, it will not be sufficient for maturation/activation of DCs. You may want to use it in combination with other DC maturation agents such as a cytokine/TLR-L cocktail (containing LPS, TNF-a etc).
As Mengyong suggested, cell surface-bound CD40L would also work better than the soluble form.
There is a literature on using monoclonal cd40 agonist antibodies to icense DCs to produce inflammatory response. I think it is more cost effective and consistent than using recombinant cd40L which would have potential stability issues over time as you store it. How are you measuring il12? Intracellular cytokine by FACS? Qpcr? ELISA? P70 can be tricky. Most easy to measure increases in p40 but then you are missing the p35 subunit but still is an accepted way to measure changes in 12.
A multimeric form of CD40L will give you better results if you do not want to use bound ligand. Quality does make a huge difference in how much stimulation you will get.
Thanks for your answers and time! Just about to try out Miltenyi's multimeric cd40L.... Will see how that works. Regards!
The anti-CD40 antibody, mab89, is by far the best activator of human cells, whether B cells or dendritic cells.
Regarding CD154 (CD40L), the trimeric form is essential, and it is the same for binding to the surface of the cell membrane, which is very far more effective in stimulating the human and animal DC.
The disadvantage is that if the stimulation with a soluble form almost does not work, it is essential to fix the MAB89 with a cell CD32-L.
For the use of membrane-bound CD154, irradiation of the presenting cell can alter the activity of trimère.Pour solve this problem we freeze dried the cell, and obtained the same results as with the MAB89 fixed, and it in many animal species.
Excellent results were obtained: for DC maturation, isotype switching of B cells, the proliferation of B cells and DC cells, the interaction between B-T-DC ...
The previous comments are right in that you have to have multimeric CD40L (at least trimeric) for good activation signals. You might consider to multimerize your CD40L by attaching it to beads.
Because you want to measure IL-12p70 consider to add IL-4, GM-CSF and IFN-gamma during your stimulation. This will drastically increase the IL-12p70 production of mouse and human DCs (for more info see: PMID 10993913
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