Violet-excited fluorochromes are becoming more commonly used in polychromatic flow cytometry experiments. However, violet-excited fluorochromes with emissions longer than 450 nm have been shown to produce small signals relative to the autofluorescent background, usable only on densely expressed antigens, and are sometimes excited by a 488 nm argon ion laser. We have developed a novel violet-excited organic fluor, Pacific Orange™ dye, which has an emission maximum at 551 nm and which is not excited by 488 nm light. Pacific Orange dye is at least twice as bright as the other green emitting violet excitable dyes, Cascade Yellow™ dye and Alexa Fluor® 430 dye. Pacific Orange dye (585/42 nm bandpass filter) can be used for two color immunophenotyping with Pacific Blue ™ dye (450/50 nm band pass filter) with minimal compensation. Data is shown comparing a human CD4/CD8 combination using a direct antibody conjugate with a Zenon® labeling reagent bound to a primary antibody. CD45 antigen is easily resolved with Pacific Orange dye, allowing CD45/SSC gating of leukocytes using violet excitation. Pacific Orange and Pacific Blue dyes can be paired with the violet-excited Fixable Aqua dead cell stain (525/50 nm bandpass filter) to exclude dead cells from immunofluorescence staining. (Figure 1) Finally, a five-color human peripheral blood leukocyte panel is shown using only violet excitation, and pairing Pacific Orange anti-CD8 and Pacific Blue anti-CD4 with Qdot® 605, Qdot 655, and Qdot 705 nanocrystal streptavidin conjugates used sequentially with biotinylated anti-CD19, anti-CD3, and anti-CD56. (Figure 2) Pacific Orange dye provides a tool to transfer detection of abundant target antigens from 488 nm excitation to the violet laser, enabling the use the 488 laser for another marker. In addition, the use of multiple violet-excited dyes can enable the detectection of five or more additional markers to standard laser combinations for greater multiplexing in polychromatic flow cytometry.

Figure 1.
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).
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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).

Figure 1.
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).
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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).

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Figure 2.
Figure 2. Five-color immunophenotyping of human leukocytes with Pacific Orange dye, Pacific Blue dye and three Qdot nanocrystal streptavidin conjugates using violet excitation. The Qdot nanocrystal staining was done with sequential staining and washing with biotinylated primary antibodies and streptavidin conjugates.
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Five-color immunophenotyping of human leukocytes with Pacific Orange dye, Pacific Blue dye and three Qdot nanocrystal streptavidin conjugates using violet excitation. The Qdot nanocrystal staining was done with sequential staining and washing with biotinylated primary antibodies and streptavidin conjugates.

Figure 2.
Figure 2. Five-color immunophenotyping of human leukocytes with Pacific Orange dye, Pacific Blue dye and three Qdot nanocrystal streptavidin conjugates using violet excitation. The Qdot nanocrystal staining was done with sequential staining and washing with biotinylated primary antibodies and streptavidin conjugates.
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Five-color immunophenotyping of human leukocytes with Pacific Orange dye, Pacific Blue dye and three Qdot nanocrystal streptavidin conjugates using violet excitation. The Qdot nanocrystal staining was done with sequential staining and washing with biotinylated primary antibodies and streptavidin conjugates.

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Topics:
dyes, flow cytometry, immunophenotyping, streptavidin, antibodies, lasers, antigens, cd45 antigens, argon lasers, cd19 antigens

Disclosures: Authors are employees of Invitrogen Corporation.; Authors have minor holdings and options in Invitrogen.; Funding as part of employment at Invitrogen.

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2006, The American Society of Hematology
2006
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