FRET-based Ca2+ measurement in B lymphocyte by flow cytometry and confocal microscopy.

Upon the B cell antigen receptor (BCR) ligation Ca(2+) mobilization is induced, which is essential for activation of downstream signaling molecules such as MAP kinase. Although synthetic fluorescent chelators such as Fluo-4 and Indo-1 are widely used for Ca(2+) measurement upon BCR ligation, they are leaked or unfavorably localized into some organelles with time post loading. To solve these problems, we introduce a genetically encoded fluorescent indicator cameleon which is a fluorescence resonance energy transfer (FRET)-based indicator comprising two fluorescent proteins (CFP and YFP) and two Ca(2+)-responsive elements (a variant of calmodulin (CaM) and a CaM-binding peptide). Here, we demonstrate that cameleon as well as a conventional synthetic Ca(2+) indicator enables Ca(2+) measurement by flow cytometry clearly upon BCR ligation. In addition, confocal microscopy analysis allows us to detect cameleon-based Ca(2+) mobilization in a single cell upon BCR ligation.