FRET causing misleading signal from fluorescein excited by the violet laser in flow cytometry.

Multiple immunolabeling introduces high risks of interferences between fluorescences. As an example, in analyzing T cell clonality, we recently reported a fluorescence resonance energy transfer (FRET) effect providing an unexpected signal on B770 (PE-Cy7) detector, on the Vβ-PE positive CD3 APC-Alexa750+ T cell subsets. Here, we report another FRET effect produced by the violet laser in Vβ-FITC positive CD3-Pacific Blue (PB) T cells providing signal on V550 (Krome Orange; KrO) detector.

Fluorescent energy transfer causing misleading signal in multicolor flow cytometry.

Multiple immunolabeling introduces high risks of interferences between fluorochromes. In an intend to analyze T cell clonality using CD3-APC Alexa750, CD4-Pac Blue, CD8-Krome Orange, CD56-PE-Cy7 and Vbeta clonotypes FITC and PE, we repeatedly observed a clear, unexpected signal on B770 (PE-Cy7) detector on the Vb subset mimicking a lymphoproliferative disorder. The aim of this study was to identify and prevent this source of artifact.