Estimation of the number of CD 154 molecules in membrane extracts used as a source of CD 40 stimulation of human B lymphocytes

Article history: Received 20 November 2008 Received in revised form 14 January 2009 Accepted 19 March 2009 Available online 27 March 2009 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 CD154transfected 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. © 2009 Elsevier B.V. All rights reserved.