Ducas Eric, Dussault Nathalie, Roy Annie, Dumont Nellie, Néron Sonia
Héma-Québec, Ingénierie Cellulaire, Recherche et Développement, Québec (Québec), Canada.
J Immunol Methods. 2009 May 31;344(2):133-7. doi: 10.1016/j.jim.2009.03.009. Epub 2009 Mar 27.
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.
CD40与CD154的相互作用用变阻器来解释比用开关更合适，其强度变化在初次和/或二次体液免疫反应后B淋巴细胞活化的调节中发挥作用。CD40与CD154的相互作用通常在使用CD154+贴壁细胞的共培养模型中进行研究，在进行蛋白质或基因分析时这可能会有问题。使用从转染了CD154的细胞制备的膜提取物可以消除由污染饲养细胞的存在引起的可能干扰。鉴于CD154对靶B细胞的剂量反应效应，使用可溶性膜时测量CD154的量很重要。我们在此报告一种基于细胞分析的简单方法，用于估计膜提取物中CD154分子的相对数量，从而使人B细胞活化水平具有可重复性。
