Estimation of the number of CD154 molecules in membrane extracts used as a source of CD40 stimulation of human B lymphocytes

The CD40–CD154 interaction is better exemplified by a rheostat than by an on–off switch, and variations in its intensity can play a role in the regulation of B lymphocyte activation following primary and/or secondary humoral immune response. The CD40–CD154 interaction is often studied in co-culture models using CD154+ adherent cells, which can be problematic when performing protein or gene analyses. The use of membrane extracts prepared from CD154+-transfected cells can eliminate possible interferences caused by the presence of contaminating feeder cells. Given the dose–response effect of CD154 on target B cells, it is important to measure the amount of CD154 when using soluble membranes. We hereby report a simple method, based on cytometry analysis, to estimate the relative number of CD154 molecules in membrane extracts, allowing reproducibility in human B-cell activation level.