Authors: G.M. Buller, Y-Z Zhang and W.L. Godfrey
Affilation: Invitrogen, United States
Pages: 575 - 578
Keywords: quantum dots, flow, tandem dyes
Qdot® nanocrystals provide fluorescent labels that can be excited with UV or violet light sources, but can also be used with longer wavelength excitation. In flow cytometry, investigators achieve greater multiplexing of cellular markers by using tandem fluors, long-wavelength organic dyes coupled to fluorescent donor proteins, phycoerythrin (RPE) or allophycocyanin (APC), to enable far red emission using 488 and 633 nm laser excitation. Tandem fluors suffer from poor stability, batch variability, and donor dye spectral bleedthrough. Qdot® nanocrystals have long Stokes shifts and relatively narrow emission peaks. Even with sub-optimal excitation at 488 and 633 nm, they provide better population resolution than is achieved with most tandem fluors. For example, Qdot®705- and Qdot®800-secondary antibody reagents allow more effective resolution of CD4+ populations in human blood than RPE and APC tandem fluors in far red emission regions. No emission is observed in the tandem fluor donor dye region, but fluorescence in the target emission region is observed with excitation off every laser. Nanocrystal reagents resolve tandem fluor issues with stability and donor bleedthrough, but require cross-laser correction. Qdot® nanocrystals can be used in place of tandem fluors in antibody staining, allowing detection of more targets over the same spectral range.