New dyes for highly sensitive analysis of cells in immunofluorescence applications

Abstract

The potential of flow cytometry and fluorescence light microscopy in the analysis of complex biological systems is still limited by the range of bright fluorochromes. The sensitivity of fluorescent probes in cell staining is usually limited by several factors including i) the ability of the fluorochrome to absorb energy and convert it into fluorescence light, ii) the extent to which the intrinsic autofluorescence of the system interferes with the fluorochrome emission, and iii) for certain applications, the resistance of the fluorochrome to fixation agents. Here we present a new set of fluorescent dyes, VB515 and VB667, which are detected in the channels of flow cytometers and fluorescence microscopes. We demonstrate that these fluorescent molecules are superior to commercially available alternatives, e.g., FITC, Alexa Fluor® 488, VioBright™ FITC, BD™ Horizon Brilliant™ Blue 515, or Alexa Fluor 647, and can even exceed the fluorescence signal of Allophycocyanine (APC)- and Phycoerythrin (PE)-conjugated antibodies. While offering higher brightness than APC- and PE, VB515 and VB667-conjugated antibodies also provide higher stability towards fixation agents in comparison to their protein counterparts. They are thus ideally suited for applications requiring high sensitivity for the detection of especially low expressed cell markers. With these new fluorochromes a multicolor panel and a gating strategy has been developed for the flow analysis of regulatory T (Treg) cells derived from PBMC and lysed blood. Treg cells play a crucial role in the suppression of non-specific or excessive immune responses. Therefore, the detailed characterization of these cells and their function is of great interest for basic and translational research.