StarBright Violet 475 (SBV475) Dye is the latest addition to the StarBright Dye range of bright fluorescent nanoparticles specifically designed for flow cytometry. It is excitable by the violet 405 nm laser and emits at 479 nm, making it easily detectable using standard filters available on most instruments.
Fig. 1. Excitation and emission spectra for SBV475. The dye is excited maximally by the violet laser (dotted line) with minimal excitation by other lasers. The emission profile is narrow to reduce spillover into neighboring filters.
StarBright Violet 475 Dye is also compatible with spectral flow cytometry and has a unique profile, allowing incorporation into new and existing flow cytometry panels, giving greater flexibility and increasing multiplex capabilities. The unique profile of SBV475 allows you to combine this dye with StarBright Violet 515 (SBV515) Dye and Brilliant Violet 510 (BV510) in a panel. The full spectral profile is shown below (Figure 2).
Fig. 2. Dye signature of SBV475. The full emission spectrum of StarBright Violet 475 Dye showing the emission at all wavelengths. Data was collected on the Cytek Aurora Flow Cytometry System using SpectroFlo Software.
SBV475 Dye has been designed to be bright with narrow excitation and emission, making it a great choice for inclusion into multicolor panels. It is up to 1.5x brighter than dyes with similar excitation and emission profiles (Figure 3), allowing detection of rare and low antigen density populations, removal of lineage positive cells, and great separation from negative cells in all multicolor panels.
Fig. 3. Brightness comparison and spectral properties. Human peripheral blood was stained with CD4SBV475 (MCA1267SBV475) (red) or CD4BV480 (blue). Both antibodies were titrated prior to use to determine the optimal concentration. SBV475 stain index was 1.5x that observed for Brilliant Violet 480 (BV480).
Fig. 4. Experimental flexibility. Human peripheral blood was stained with CD4SBV475 (MCA1267SBV475) in PBS 1% BSA, bovine calf serum containing Staining Buffer (BUF073), or Brilliant Stain Buffer with no detectable differences.
No special staining buffer is required. SBV475 Dye has been tested in most common staining buffers and special staining buffers for polymer dyes, with no drop in performance, so it can be used in new or existing panels, with no change to your experimental protocol (Figure 4).
Furthermore, SBV475 Dye is very stable when stored at 4oC and is not susceptible to photobleaching by ambient light. Staining does not deteriorate with fixation with either formaldehyde or alcohol based fixatives and fixative/permeabilization reagents.
Like all StarBright Dyes, SBV475 has high lot-to-lot reproducibility, so you can expect the same high-quality staining regardless of the lot purchased.
StarBright Violet 475 Dye is compatible with all common fluorophores such as organic fluorophores, protein fluorophores, polymer dyes, and other StarBright Dyes in multicolor panels, without the requirement for a special staining protocol or special staining buffer. Examples of staining with other dyes, including StarBright Dyes, can be seen in Figure 5. If you are using multiple dyes that require a special buffer for optimal staining, we recommend using that buffer, as StarBright Dye performance will not be affected by buffer choice.
Fig. 5. Examples of SBV475 Dye staining. Human peripheral blood was stained with A, CD20PE (MCA1710PE) and CD19SBV475 (MCA1940SBV475); B, CD3A700 (MCA463A700) and CD27SBV475 (MCA755SBV475); C, CD3SBB700 (MCA463SBB700) and CD4SBV475 (MCA1267SBV475); and D, CD3BV711 (BD) and CD4SBV475 (MCA1267SBV475). All staining was performed at 4oC in PBS containing 1% BSA after blocking with 10% human serum. Cells were analyzed on the ZE5 Cell Analyzer.
For more information on how the StarBright range can fit into your flow cytometry experiments, please take a look at our Starbright Dyes page.
For more information about fluorophores and immunophenotyping, refer to our flow cytometry resources.
Click on the links below to find out more in-depth information on each topic and view our popular flow cytometry basics guide.