Multicolor Flow Cytometry

BD Biosciences continues to expand the options for multicolor flow cytometry through the development of a new BD Horizon™ brand dye for flow cytometers equipped with blue (488-nm), green (532-nm), or yellow-green (561-nm) lasers. The BD Horizon™ PE-CF594 dye has been developed by BD Biosciences specifically to have improved brightness and spectral characteristics over current dyes used for the PE-Texas Red detector.

A new choice for the blue, green, and yellow-green lasers

BD Horizon PE-CF594 is an analog of PE-Texas Red, which has improved performance in multicolor panels. Because PE has maximum excitations at 496 nm and 564 nm (Figure 1), BD Horizon PE-CF594 can be excited by blue, green, and yellow-green lasers. With a maximum emission at 612 nm, BD Horizon PE-CF594 is readily compatible with standard PE-Texas Red filter sets available for many BD FACS™ brand cytometers, including the BD FACSCanto™ II flow cytometer, BD FACSAria™ cell sorter platform, BD™ LSR cell analyzer platform, and the BD FACSVerse™ flow cytometer.

Figure 1. Absorption and emission spectra

Figure 1. Absorption and emission spectra.

Ex Max: 496 nm and 564 nm, Em Max: 612 nm.

Improved brightness and consistent spillover values

BD Horizon PE-CF594 reagents maximize choice and flexibility by providing an additional bright dye that can be used in multicolor panels. BD Horizon PE-CF594 is brighter than other dyes currently offered for this detector with similar or lower background (Figure 2 and Table 1). Due to its emission spectra, PE-CF594 has significantly less spillover into the PerCP and PE-Cy™7 detectors when compared to PE-Alexa Fluor® 610 (Table 1). Additionally, BD Horizon PE-CF594 reagents exhibit consistent spillover values between lots and specificities, minimizing the need for lot specific compensation controls (Table 2). These improved spectral characteristics make BD Horizon PE-CF594 an optimal choice for multicolor panels.

Figure 2. Staining comparison of BD Horizon PE-CF594, ECD and PE-Alexa Fluor® 610:

Figure 2. Staining comparison of BD Horizon PE-CF594, ECD, and PE-Alexa Fluor® 610.

A. CD3: Lysed whole blood stained with human CD3 conjugated to BD Horizon PE-CF594, PE-Texas Red, ECD, or PE-Alexa Fluor® 610, run on a BD LSR II system (using a 610/20-nm filter and the 488-nm laser). All conjugates were run at the manufacturer’s recommended concentration.

B. CD4: Lysed whole blood stained with human CD4 conjugated to BD Horizon PE-CF594, ECD (PE-Texas Red, Beckman-Coulter), or PE-Alexa Fluor® 610, run on a BD LSR II system (using a 610/20-nm filter and the 488-nm laser). All conjugates were run at the manufacturer’s recommended concentration.

Table 1. Stain Index and spillover comparison

Table 1. Stain Index and spillover comparison.

Lysed whole blood stained with human CD3, CD4, or CD19 conjugated to BD Horizon PE-CF594, ECD, PE-Texas Red, or PE-Alexa Fluor® 610, run on a BD LSR II system (using a 610/20-nm filter and the 488-nm laser). All conjugates were run at the manufacturer’s recommended concentration.

Table 2. Spillover value comparison between specificities

Table 2. Spillover value comparison between specificities.

Lysed whole blood from a single donor stained with human CD3, CD4, CD8, CD14, CD16, CD19, CD20, or CD28 conjugated to BD Horizon PE-CF594. All conjugates were run side by side on a BD LSR II system (using a 610/20-nm filter and the 488-nm laser), at their optimal concentration.

Compatible with standard surface and intracellular staining protocols

BD Horizon PE-CF594 is compatible with standard buffers used in surface and intracellular staining protocols, including BD Cytofix™ fixation buffer/BD Perm/Wash™ buffer, Human FoxP3 Buffer Set, BD Phosflow™ fixation buffer I/BD Phosflow™ perm/wash buffer I, BD Phosflow™ perm buffer II, BD Phosflow™ perm buffer III, and BD Phosflow™ perm buffer IV. The following plots show CD3 and CD19 BD Horizon PE-CF594 staining using various staining protocols. Note that staining intensity under various fixation/permeabilization conditions is variable between proteins, so buffer compatibility is a function of the target protein, fluorochrome, and antibody used.

Figure 3. Example of BD Horizon PE-CF594 staining using various protocols

Figure 3. Example of BD Horizon PE-CF594 staining using various protocols.

Histogram overlays of human CD3 or CD19 staining using various staining protocols: live-cell stain, cell staining post fixation/permeabilization, or cell staining prior to fixation/permeabilization.

Maximize the power of your multicolor experiments

BD Horizon PE-CF594 allows you to take full advantage of your instrument capabilities, increasing the power of your experiments. Using BD Horizon PE-CF594 in combination with other dyes offered by BD Biosciences, allows you to detect 10 fluorescence parameters from a single sample.

Figure 4. Example data from a 10-color TBMNK panel

Figure 4. Example data from a 10-color TBMNK panel.

Example data of BD Horizon PE-CF594 used in a 10-color panel to phenotype T cells, B cells, monocytes, and NK cells. CD8 vs CD57 was used to look at CD8 terminal differentiation of the NK cells. PBMCs were stained with CD16 BD Horizon PE-CF594, CD3 BD Horizon™ V450, CD45 BD Horizon™ V500, CD57 FITC, CD19 PE, CD4 PerCP-Cy™5.5, CD14 PE-Cy7, CD56 APC, CD20 Alexa Fluor® 700, and CD8 APC-H7. This panel was run on a BD LSRFortessa™ system with violet (405-nm), blue (488-nm) and red (640-nm) lasers.

Figure 5. Example data from a 9-color Memory/Effector T-cell Panel

Figure 5. Example data from a 9-color Memory/Effector T-cell panel.

Example data of BD Horizon PE-CF594 used in a 9-color panel to phenotype various T-cell populations, including Terminal Effector Memory (TEM), Effector Memory (EM), and Central Memory (CM). Use Table 3 as a reference for the markers used in this panel. Lysed whole blood was stained with human CD27 BD Horizon PE-CF594, CD62L BD Horizon V450, CD4 BD Horizon V500, CD3 FITC, CCR7/CD197 PE, CD28 PerCP-Cy5.5, CD127 APC, CD45RA Alexa Fluor® 700, and CD8 APC-H7. This panel was run on a BD LSRFortessa system with violet (405-nm), blue (488-nm), yellow-green (561-nm) and red (640-nm) lasers. The PE and BD Horizon PE-CF594 reagents were excited by the 561-nm laser.

Table 3. Common Markers used for T-cell Memory

Table 3. Common markers used for T-cell memory.