B cell functional assays

Gras gras at dsvidf.cea.fr
Wed Jan 15 07:43:40 EST 1997

For assessing CD40, ICAM1 and LFA1 modulations, take care of 
having a very thin distribution and use a quantitation kit (eg 
QIFIkit from Dako). Modulations could be minor. You could perhaps 
add B7s (CD80 and 86), CD71, CD25 detection.
We have never used LPS, a friend told me it was not very efficient 
in human B cells.
SAC is rather near anti-Mu stimulation.
You can activite with anti-Mu coated beads and then perform flow 
cytometry : beads are before cells in the scattergram (ours) and 
do not keep attached during the staining.
We have no CD40L cell line, but we use G28.5 mAb from Pr E Clark 
(Seattle WA) which activates well (10mcg/ml without any 
crosslinking). Keep in mind that : (i) this mAb links an epitope 
outside the CD40/CD40L site, but is a pretty good reagent (and the 
Hybridoma is at ATCC) ; (ii) G28.5 treatment lead to high 
background if you use indirect immunostaining.
In my opinion, I would use anti-Mu and/or SAC, and CD40 mAb 
together with combinations of IL2/IL4.
For detailed assays description, read Virology 1996, 220:309-319, 
which well describes our type of approach.

Hopping it helps, sincerely

Gabriel Gras

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