Multicolor Flow Cytometry

Sample Data

BD Biosciences continues to expand the options for multicolor flow panel design with the development of a completely new BD Horizon™ brand dye for the violet laser. The BD Horizon™ V500 dye has been specifically engineered to improve brightness over Pacific Orange™ and reduce spectral overlap into the FITC channel compared to AmCyan.

BD Horizon V500 is a completely novel organic dye structure that has been specifically engineered to have optimal spectral characteristics for use with the violet laser. BD Horizon V500 is optimally suited for use with BD FACS™ brand flow cytometers equipped with violet lasers, including the BD FACSCanto™ II flow cytometer, BD FACSAria™ cell sorters, and the BD™ LSR cell analyzer family.

With a maximum excitation at 415 nm and an emission peak at 500 nm, BD Horizon V500 is readily compatible with the standard filter sets on BD Biosciences FACS brand flow cytometers equipped with violet lasers and is an optimal alternative to Pacific Orange™ and AmCyan.

Improved Brightness over Pacific Orange™

CD4 BD Horizon V500 comparison to Pacific Orange™

Freshly isolated human lymphocytes stained with CD4 (RPA-T4) conjugated to BD Horizon V500, Pacific Orange™, or AmCyan. Samples run on a BD FACSCanto II flow cytometer. BD Horizon V500 and AmCyan data collected with a 510/50 nm, 502 LP filter. Pacific Orange™ data collected with a 585/42 nm filter. The plots have been superimposed to show signal comparison.

CD8 BD Horizon V500 comparison to Pacific Orange™

Freshly isolated human lymphocytes stained with CD8 (RPA-T8) conjugated to BD Horizon V500 or Pacific Orange™. Samples run on a BD FACSCanto II flow cytometer. BD Horizon V500 data collected with a 510/50 nm, 502 LP filter. Pacific Orange™ data collected with a 585/42 nm filter. The plots have been superimposed to show signal comparison.

BD Horizon V500 stain index comparison to Pacific Orange™

Freshly isolated lymphocytes stained with human CD4, CD8, CD19, or CD45 conjugated to BD Horizon V500, Pacific Orange™, or AmCyan. Samples run on a BD FACSCanto II flow cytometer. BD Horizon V500 and AmCyan data collected with a 510/50 nm, 502 LP filter and Pacific Orange™ data collected with a 585/42 nm filter.

Reduced spillover into the FITC channel

FITC spillover comparison

Lysed whole blood stained with CD4 (RPA-T4) conjugated to BD Horizon V500 or AmCyan. Run on a BD FACSCanto II flow cytometer. Spillover detected in the FITC channel.

% spillover into FITC channel comparison

Comparison of lysed whole blood stained with CD4, CD19, or CD45 conjugated to V500 vs AmCyan. Experiment run on a BD™ LSR II flow cytometer, with spillover detected in the FITC channel.

BD Horizon V500 opens up more options for multicolor flow assays and is an optimal accompaniment to BD Horizon V450

Hu CD45 PerCP vs SSC

Lysed whole blood stained with CD3 (UCHT1) BD Horizon V450, CD4 (RPA-T4) BD Horizon V500, CD8 (SK1) APC, CD16 (3G8) FITC, CD19 (HIB19) PE, and CD45 (2D1) PerCP. Experiment was run on a BD FACSCanto II flow cytometer.

Hu CD4 BD Horizon V500 vs CD3 BD Horizon V450

Lysed whole blood stained with CD3 (UCHT1) BD Horizon V450, CD4 (RPA-T4) BD Horizon V500, CD8 (SK1) APC, CD16 (3G8) FITC, CD19 (HIB19) PE, and CD45 (2D1) PerCP. Data shown gated on lymphocytes. Experiment was run on a BD FACSCanto II flow cytometer.

Hu CD16 FITC vs CD3 BD Horizon V450

Hu CD19 PE vs CD3 BD Horizon V450

Hu CD8 APC vs CD3 BD Horizon V450

Ms CD4 BD Horizon V450 vs CD8 BD Horizon V500

Splenocytes from a C57/BL6 mouse were stained with CD3 (145-2C11) FITC, CD4 (RM4-5) BD Horizon V450, CD8 (53-6.7) BD Horizon V500, CD45 (30-F11) PerCP, and NK1.1 (PK136) PE and run on a BD LSR II cell analyzer. Data shown gated on CD45-positive splenocytes.

Ms NK1.1 PE vs CD3 FITC

Ms NK1.1 PE vs CD8 BD Horizon V500

Ms NK1.1 PE vs CD19 APC

Ms CD19 APC vs CD8 BD Horizon V500

Ms NK1.1 PE vs CD4 BD Horizon V450

Note: When compensating dyes in this spectral range (such as BD Horizon V500 and AmCyan), the most accurate compensation can be obtained using single-stained cellular controls. Due to spectral differences between cells and beads in this channel, using BD™ CompBeads can result in spillover errors for BD Horizon V500 and AmCyan reagents. Therefore, the use of BD CompBeads or BD CompBeads Plus to determine spillover values for these reagents is not recommended. Different BD Horizon V500 reagents (eg, CD4 vs CD45) can have slightly different fluorescence spillover. Therefore, it may also be necessary to use clone-specific compensation controls when using these reagents.