RealBlue 705: A Next-Generation Upgrade for Cleaner, Brighter Flow Cytometry

When it comes to multicolor flow cytometry, every fluorochrome choice affects your data quality. Small changes in brightness or spectral spillover can determine whether your rare population is crisp or lost in the noise. That’s where RealBlue 705 (RB705) comes in — BD’s modern alternative to the long-standing PerCP-Cy5.5.

Whereas PerCP-Cy5.5 works reliably in many standard panels, it tends to contribute more spillover into adjacent channels and can complicate compensation, especially when detecting low-abundance markers.

Key Advantages of RealBlue 705

1. Brighter signal for low and medium expression markers
RB705 delivers significantly higher fluorescence intensity compared to PerCP-Cy5.5. This makes it ideal for detecting markers with subtle expression levels — such as regulatory or activation markers that often sit near the detection threshold.

2. Cleaner spectra and reduced spillover
RB705’s emission profile is narrower and better confined, leading to less spectral overlap into neighboring channels. In practice, this means cleaner separation between populations and reduced compensation complexity, particularly when combining multiple far-red dyes.

3. Minimal cross-laser excitation
One of PerCP-Cy5.5’s drawbacks is partial excitation from the 561 nm yellow-green laser, which can cause unwanted background in other detectors. RB705 shows minimal cross-laser excitation, maintaining signal purity even in advanced multi-laser cytometers.

4. Optimized for complex panels
Because RB705 introduces less spillover spread, it simplifies panel design and allows users to add more parameters without sacrificing resolution. This is especially valuable for high-parameter or spectral cytometry workflows.

5. Compatible with fixation and permeabilization buffers
RB705 maintains stability through common fixation and permeabilization processes — making it suitable for intracellular and nuclear staining workflows where PerCP-Cy5.5 performance can be inconsistent.

Where PerCP-Cy5.5 Still Holds Its Ground

PerCP-Cy5.5 remains widely available and familiar to most cytometrists. It performs well for bright, surface markers and in simpler 8–12-color panels where spillover is less of an issue. Its widespread use means compensation settings and reference controls are well documented. However, for modern 18+-color configurations or low-signal markers, its brightness and spectral footprint can become limiting.

Practical Tips for Users

• Upgrade gradually: Start by swapping RB705 into one marker position currently labelled with PerCP-Cy5.5 to compare performance side-by-side.

• Re-titrate antibodies: RB705’s higher brightness may require lower antibody concentrations to prevent oversaturation.

• Validate compensation: Even with reduced spillover, new fluorochromes should always be individually compensated.

• Check your filter set: RB705 emits around 705 nm — make sure your far-red filter and PMT are properly configured to capture it efficiently.