From: Simon Monard (Simon_Monard@adarc.org)
Date: Mon Dec 30 1996 - 09:22:15 EST
Hi Keith 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
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