I have never tried to stimulate B-cells with anti CD40 but have stimulated them
with plenty of other things. To produce a calcium flux you probably have to
cross-link the CD40 molecules, so add CD40 mAb, wash, then follow with an
unlabelled goat anti mouse Ig or something. I would suspend your cells in Hanks
with calcium and magnesium dunno about IL-4, we never needed it for our
experiments, let your cells "rest" after any separation before trying to get a
calcium flux
Calcium ionophore is used to make sure your detection system is working and
sometimes to get the "maximum" response when "trying" to calculate actual
intracellular calcium concentrations. It reflects no physiological process.
Cross linking with a second immunoglobulin does pretty much the same job as
beads. B-cells are very sensitive to cross linking of their surface IgM, when
using a second antibody make sure it doen't cause a flux by cross reacting with
the B-cell surface Ig
I would be very careful about separating your cells in any way prior to doing a
calcium assay, I would probably ficol to get rid of red cells platelets and
neutrophils, then stain all the cells you DO NOT want and gate them ut during
the calcium assay, ie stain T-cells and monocytes with PE or something.
Staining your B-cells would complicate the calcium assay as when you crosslink
with your second antibody you would be co-ligating the marker you used for your
selection. That could have either an up or down regulatory effect.
To get consistant results with these assays you must prepare your cells in a
very consistant way and be gentle with your cells.
Good luck
Simon Monard
Aaron Diamond Center
New York
NY 10016