What is your goal?

want to stimulate B-cell subtypes? Immortalize PBMC? monitoring the production of antibodies? or clone B cells producing specific antibodies?

The use of soluble CD40L or attached to the beads is quite inefficient, due to the fact that the trimeric form of the ligand is required, and is not present in a production of bacterial origin.

You can use the CD40 system, where the CD40 antibody is associated with EBV. You can also use the cells transfected with CD40L.

But now, the best performance is obtained with the HuBBB kit that combines these two modes of stimulation.

Hi,

I want to see the activation of the NFkB pathway as well as proliferation in CD40L-stimulated B cells isolated from PBMC as well as in LCL (so immortalized with the EBV). We have a cell line that express the CD40L but for simplicity of experiment in the beginning and to avoid co-cultures, I would like to try first stimulation with CD40L by itself.

Thanks for your answer

If it is the murine L929 cell line transfected with CD40 ligand from Schering-Plough, this line works very well because it expresses the trimeric form with a good level, Of course, when it is not derived. You should check the expression level of this cell line with an anti-CD154 antibody by FACS analysis.

After irradiation, this line is highly effective to stimulate B cells after PBL, but beware, CD40L can boost monocytes, MoDC and pDC which are also present in peripheral blood.

In the past we have shown that the irradiated line had no feeder effect by itself, but slightly toxic if too heavily irradiated.

Using an antibody or CD40L(CD154) beads will only give a week CD40 stimulation. We currently use the MegaCD40L from ENZO with excellent results. It is a construct in which two trimeric CD40 ligand molecules are artificially linked via the collagen domain of Adiponectin/ACRP30/AdipoQ. This construct very effectively simulates the natural membrane-assisted aggregation of CD40L in vivo.

Moreover, we use a mouse myeloma cell line P3xTB.A7 stably expressing human CD154 and the non-transfected control P3x63Ag8.653 cells.

I agree with Dr Pin and Wagner. On the other hand, I have used irradiated HL60 cells at a 1:1 ratio with excellent results for human B cell stimulation.

Dr Wagner,

Is the  P3xTB.A7 cell line available (buy/licence) ? I've tried MegaCD40L with B cell lymphoma cell lines but it doesn't work (up to 10 µg/ml). So a CD40L expressing cell line could help us.  Thanks.

The mouse myeloma cell line P3xTB.A7 stably expressing human CD154 and the

non-transfected P3x63Ag8.653 control cells were kind gifts of Prof. R. Kroczek

(Robert-Koch-Institute, Berlin): [email protected]. Phone (work): +49 30 4547-2450.

In addition some groups use CD154 expressing baby hamster kidney (BHK) cells.

10 µg/ml MegaCD40L is pretty much, should work ... normally we use 200 ng/ml to stimulate endothelial cells. Is the CD40 receptor expressed in your B cell lymphoma cell line?

Dear Andreas,

Did you tried use MegaCD40L to culture sorted single murine B cell?

thanks

Yuxi

Dear Yuxi, so far we used primary human umbillical vein endothelial cells(HUVEC) and isolated human monocytes (negative selection wuth Milteny beads) for MegaCD40L stimulation and it worked very well. We also used MegaCD40L (200 ng/ml) from Enzo for stimulation of murine endothelial cells in situ (carotid artery) and it also worked well. If you compare the sequence between mouse and humanyou will find a homology of approx. 85%. There is also a murin recombinant CD40L available but not as "mega" (molecule of two linked trimeris) ... you can buy a kind of enhancer, but we did not try that because the human MegaCD40L also worked in the murine system.

Thank you, Andrea,

  For my understanding, you use primary HUVEC as feeder cells, and they will produce some components for B cell growth and die sooner. You are the expert in this field, and I just wonder can I directly culture single sorted memory B cell without Feeder cells? I can buy the megaCD40L and add them in to medium, and other cytokine, IL2, IL21, I think.

 I am a new guy in this field and we do not have any Gamma ray resource...

many thanks

Yuxi

No, we do not use HUVEC as feeder cells for B cell culture. I am not an B cell expert but I am sure there are some publications availabe about culture conditions. For the culture of isolated monocytes you must have M-CSF or GM-CSF in your medium.

I saw some people just use irradiated CD40L-3T3 cells, IL2 and IL21 for the sorted mouse memory cells, does 3T3 cells produce some M-CSF or GM-CSF?

I am quite sure that 3T3 do not produce GM-CSF or M-CSF.You have to add it as a supplement to your culture medium.