B cell functional assays

Keith Bahjat Kbahjat at nwu.edu
Sat Jan 11 02:42:44 EST 1997



Gras <Gras at dsvidf.cea.fr> wrote in article <32D29B03.7FE3 at dsvidf.cea.fr>...
> The success depends on the what you're looking for : PMA+Iono 
> activates very well but kills a lot of cells, SAC is more specific 
> and kills less. IL4 is a good survival signal, especially after 
> CD40 triggering. In our experiments, mixed IL4 and IL2 are very 
> good.
> 
> Tell precisely what you want, we'll help.
> 

What I hope to come up with is a combination of two or three assays which
will activate B-cells via different mechanisms, thus allowing me to
hypothesize about  their ability to react to stimuli in vivo. This would be
used in combination with routine activation markers to first assess the in
vivo state of the cells, and then assess their functional abilities as
compared to normal controls. Once stimulated, cells could be analyzed for
upregulation of CD40, ICAM-1, LFA-1, proliferative ability, calcium flux
(with indo-1), etc.

I am thinking of using LPS for a T-ind. type 1 stimulation (not sure yet
which cytokines I also would need), and anti-Ig-dextran + IL2 for a T-ind.
type 2 activation.A third possibility using a cell line transfected to
express CD40, in addition to cytokines, to simulate a T-dependent response,
is waiting for more info.

Is SAC supposed to simulate a T ind type 1 response?

When you activate with anti-Ig or anti-Ig-dextran on polyacrylamide beads,
do the beads interfere with light scatter patterns for flow cytometry?

Any ideas for CD40 crosslinking besides using transfected cells? Any cell
lines that already express CD40L?

All the info I have is from journal and text searching, so it is very
vague. Any and all help is appreciated.

Thanks.

Keith Bahjat
Northwestern University Medical School




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