Pacific Orange dye has a maximum emission wavelength of 551 nm and is not excited by 488 nm light. Pacific Orange dyes are at least twice as bright as other green-emitting violet-excitable dyes Cascade Yellow and AlexaFluor 430. Pacific Orange Dye (585/42 nm Bandpass Filter) can be used for two-color immunotyping of Pacific Blue Dye (450/50 nm Bandpass Filter) with minimal compensation. Data comparing human CD4/CD8 combinations using direct antibody conjugates with labeled reagents bound to the primary antibody are shown. The CD45 antigen is easily resolved with Pacific Orange dyes, allowing CD45 / SSC gating of leukocytes by violet light excitation. Pacific Orange and Pacific Blue dyes can be paired with violet-excited Fixable Aqua dead cell stains (525/50 nm bandpass filters) to exclude dead cells from immunofluorescence staining. (Figure 1) Finally, only five colors of human peripheral blood leukocyte plates were displayed using violet light excitation, and Pacific Orange anti-CD8 and Pacific Blue anti-CD4 were used with Qdot 605, Qdot 655, and Qdot 705 nanocrystalline streptavidin the conjugate paired was biotinylated anti-CD19, anti-CD3, and anti-CD56. Pacific Orange Dye provides a tool to transfer the detection of rich target antigens from 488 nm excitation to a violet laser, so that the 488 laser can be used for other labels. In addition, five or more other markers can be detected using multiple violet-excited dyes to combine with a standard laser combination for greater multiplexing in multicolor flow cytometry.
Figure 1. Immunophenotyping of mixed live and heat-killed human leukocytes using Pacific Orange dye, Pacific Blue dye and the Fixable Aqua dead cell reagent. Live cell events (Fixable Aqua stain-negative) were gated in the histogram (left) for display in the CD4/CD8 scatter plot (right). (Gayle M. Buller.; et al. Blood,2006)
Pacific Orange dye is a bright orange-fluorescent dye optimally excited by the 405 nm line of the violet laser and with excitation/emission at 400/551 nm. Conjugates of this dye are strongly fluorescent even at neutral pH. Pacific Blue, Pacific Green and Pacific Orange dye conjugates can be simultaneously excited at 405 nm and emit at 455 nm, 500 nm and 551 nm, respectively, facilitating three-color analysis. We offer Pacific Orange dye conjugated to a variety of primary antibodies, secondary antibodies and streptavidin. In addition, reactive and protein labeling kits are available so that you can produce your own antibody conjugates or probes.