New violet-excitable reagents for multicolor flow applications.

We have recently added three new fluorophores-BD Horizon™ V450, BD Horizon V500, and BD Horizon V550 (V450, V500, and V550; BD Biosciences, San Jose, CA) to our existing AmCyan product, forming a group of four violet-excitable dyes from which we have produced functional antibody conjugates. These conjugates, with emission maxima that range from 450 to 535 nm, are compatible with multilaser flow cytometry (FCM) and can be used for polychromatic FCM in three-color or two-color combinations; in fact, V500 fills a spectral opening that has thus far not been exploited by other manufacturers of FCM reagents. We here report that conjugates based on BD Horizon dyes performed well within a useful sensitivity range, established by testing a representative group of violet-excitable FCM reagents currently available, and that V500 has better compatibility with FITC in multicolor applications than does AmCyan.

Use of a new violet-excitable AmCyan variant as a label in cell analysis.

Here we report a new variant of AmCyan fluorescent protein that has been specifically designed for multicolor cell analysis. AmCyan is one of the existing violet fluorochromes for use in flow cytometers equipped with a violet (405 nm) laser. It is also widely used as a label in fluorescent spectroscopy.

Control of the fluorescence of dye-antibody conjugates by (2-hydroxypropyl)-β-cyclodextrin in fluorescence microscopy and flow cytometry.

When proteins are conjugated to fluorescent organic dyes, fluorescence emission of the dye molecules is usually decreased, sometimes up to 50-70%. This quenching phenomenon has been acknowledged for decades, but as yet, there are no simple, practical methods to control the fluorescence of dyes conjugated to proteins, especially for dyes conjugated to immunoglobulins. Here, we report that the addition of (2-hydroxypropyl)-β-cyclodextrin (HPβCD) to dye-antibody conjugates can increase fluorescence up to 2.

3-Carboxy-6-chloro-7-hydroxycoumarin: a highly fluorescent, water-soluble violet-excitable dye for cell analysis.

In our search for new violet-excitable dyes with improved photophysical and photochemical properties, we examined several halogen-substituted hydroxycoumarins and found that chlorinated derivatives are at least as bright as their fluorinated analogs. A monochlorinated hydroxycoumarin was found to have a high quantum yield (approximately 0.98), and human leucocyte-specific monoclonal antibodies (CD3, CD4, and CD45) conjugated with this dye exhibited reliable performance in flow cytometry assays.

Quantum dots in flow cytometry.

The development of new fluorophores has experienced a tremendous advance over the last two decades. The unique photophysical properties of quantum dots (QDs), such as their large Stokes shifts and exceptional brightness, make them attractive probes in flow cytometry applications. In this chapter, the spectral overlap and the fluorescence intensity of a single Qdot nanocrystal species (Qdot-655) was investigated in the context of a panel containing conventional fluorophores.