Expand Your Multicolor Capacity With the Pacific Green™ Dye

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Violet light–excitable fluorophores play a critical role in the expansion of multiparametric flow cytometry, enabling more markers to be detected in a single sample. With the advancement of these fluorescent dyes has come the widespread availability of flow cytometers equipped with solid-state violet laser diodes, which are both affordable and reliable. The expansive Molecular Probes® dye portfolio supports multicolor immunophenotyping by flow cytometry and includes a variety of dyes compatible with the 405 nm spectral line of the violet laser. The newest fluorophore in the violet light–excitable Pacific dye family is Pacific Green™ dye, a small-molecule organic dye with bright green fluorescence that fills the spectral gap between the Pacific Blue™ and Pacific Orange™ fluorophores.

Pacific dyes are all compatible with violet laser excitation

Pacific Green™ dye is a violet light–excitable fluorophore with excitation and emission maxima of 411 nm and 500 nm, respectively. Pacific Green™ dye joins Pacific Orange™ and Pacific Blue™ dyes, which are established violet laser–excitable fluorophores for flow cytometry. Pacific Blue™, Pacific Green™, and Pacific Orange™ dye conjugates can be simultaneously excited at 405 nm for emission at 455 nm, 500 nm, and 551 nm, respectively (Figure 1), enabling three-color analysis with a single laser line. When used for three-color immunophenotyping, these Pacific dyes require only minimal compensation and exhibit very little 488 nm cross-excitation.

Figure 1. Fluorescence excitation (dotted lines) and emission (solid lines) spectra for Molecular Probes® violet light–excitable Pacific Blue™ dye (blue lines), Pacific Green™ dye (green lines), and Pacific Orange™ dye (orange lines). The shaded area represents the Attune® emission bandpass filter (522/31) used for detecting the Pacific Green™ dye.

Expand the number of targets detected using 405 nm excitation

The development of violet light–excitable fluorophores addresses the need in multiparametric flow cytometry experiments to transfer markers off the blue (488 nm) and red (635 nm) excitation lines and onto the violet (405 nm) excitation line. By including violet light–excitable fluorophores in the experiment, researchers can free up the 488 nm and 635 nm laser lines for detection of more specialized markers, for which blue light– and red light–excitable fluorescent conjugates are more likely to be commercially available.

Figure 2 shows an example of multicolor immunophenotyping of normal human blood using four different primary antibody conjugates, each labeled with either the Pacific Blue™, Pacific Green™, Pacific Orange™, or Alexa Fluor® 488 fluorophore. In addition, Pacific Green™ conjugates can be paired with the violet light–excitable LIVE/DEAD® Fixable Violet Dead-Cell Stain (455 nm emission) or LIVE/DEAD® Fixable Yellow Dead-Cell Stain (550 nm emission) for two-color detection, or with far red–emitting Qdot® 605, Qdot® 655, or Qdot® 705 conjugates for higher-order multiplexing with violet laser excitation. When selecting cells to eliminate from further analysis, Pacific Green™ conjugates can be combined with the LIVE/DEAD® Fixable Aqua Dead-Cell Stain (525 nm emission) for use in a “dump channel”.

Figure 2. Bivariate plot displays for multicolor immunophenotyping by flow cytometry. Normal human blood was labeled with four different fluorophore-conjugated primary antibodies. (A) Lymphocyte gating with the Pacific Orange™ conjugate of anti–human CD45 antibody was used as the parent for the subsequent bivariate plots. Fluorescence of the Pacific Green™ conjugate of anti–human CD3 antibody was plotted against the fluorescence of (B) the Pacific Blue™ conjugate of anti–human CD8 antibody and (C) the Alexa Fluor® 488 conjugate of anti–human CD56 antibody. Data acquisition was performed on the Attune® Acoustic Focusing Cytometer with the violet (405 nm) and blue (488 nm) laser options and the standard event collection rate of 100 μL/min; data were analyzed using Attune® Software.

Amine-reactive Pacific Green™ dye and ready-to-use conjugates

Pacific Green™ dye is available in multiple product formats to fit most applications (Table 1). These include Pacific Green™ primary and secondary antibody conjugates that have been validated for flow cytometry, as well as Pacific Green™ streptavidin.
For protein labeling applications, we offer the Zenon® Pacific Green™ Mouse IgG1 Labeling Kit for creating direct primary antibody conjugates, as well as the amine-reactive Pacific Green™ succinimidyl ester, an efficient and easy-to-use reagent for selectively linking the dye to accessible primary amines. Pacific Green™ succinimidyl ester can also be used for fluorescent cell barcoding to facilitate high-throughput multiplex flow cytometry [1].

Table 1. Molecular Probes® Pacific Green™ conjugates and protein labeling reagents.

References

1. Krutzik PO, Clutter MR, Trejo A et al. (2011) Curr Protoc Cytom 6.31.1–6.31.15.