StarBright Dyes in Premixed Cocktails

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Overview

Building flow cytometry panels can be difficult and time consuming, especially for those new to flow cytometry. One easy option is to purchase commercially available premixed panels that have been optimized in terms of staining concentration and fluorophore selection. They are available in a range of sizes, with the most common being just 3- or 4-plex, and allow you to simply stain your cells and analyze the results, often without the need for compensation.

Easy Experiment Expansion

To test their flexibility, StarBright Dyes were added to three-color, premixed antibody cocktails, available from Bio-Rad and other vendors, following multicolor panel building best practices, without compensating the data.

Fig. 1. Example of three-color cocktail staining. Human peripheral blood was stained with the three-color premixed cocktail of CD3, CD4, and CD8. The gating strategy shows the A, single cells; B, lymphocytes C, CD3⁺ T cells and D, CD4 and CD8 T helper and T cytotoxic cells. All staining was performed at 4^oC in PBS containing 1% BSA, after blocking with 10% human serum. Cells were analyzed on the ZE5 Cell Analyzer.

When CD19 StarBright Violet 440 (SBV440) Dye was simply added at the recommended concentration, without any change to the existing protocol, the ability to identify the T cells was unaffected, and the B cell population could now also be identified (Figure 2). In this staining, we also added the live/dead marker PureBlu DAPI to  remove the dead cells.

Fig. 2. Adding SBV440 to a Bio-Rad three-color cocktail. Human peripheral blood was stained with the three-color premixed cocktail of CD3, CD4, and CD8, PureBlu DAPI (1351303), and CD19SBV440 (MCA1940SBV440). The gating strategy shows the live/dead, single cells, the lymphocytes, the CD3⁺ T cells, and CD19⁺ B cells, and finally the CD3⁺, CD4, and CD8 T helper and T cytotoxic cells. All staining was performed at 4^oC in PBS containing 1% BSA, after blocking with 10% human serum. Cells were analyzed on the ZE5 Cell Analyzer.

Cocktail Compatibility

To confirm StarBright Dyes are compatible with a variety of premixed cocktails, premixed panels from two other vendors were used and whole blood stained. StarBright Dyes were then added at the recommended concentration and whole blood retested, without changing the staining protocol or buffer, and without compensation. A live/dead cell marker was also used in each staining panel. When CD19SBV440 and CD8SBB700 were added to the panel from vendor one (Figure 3), extra T cell and B cell populations could be identified without affecting identification of the other population.

Fig. 3. Adding SBV440 and StarBright Blue 700 (SBB700) Dyes to a four-color cocktail from vendor one. Human peripheral blood was stained with A, the four-color premixed cocktail of CD3, CD4, CD127 and CD25, or B, the four-color premixed cocktail plus PureBlu DAPI (1351303) and CD19SBV440 (MCA1940SBV440). The gating strategy selected the live cells, single cells, then the lymphocytes. The CD3⁺ T cells, CD19⁺ B cells, the CD3⁺, CD4, and CD8 T helper and T cytotoxic cells, and the CD127^hi CD25^lo Treg cells. All staining was performed at 4^oC in PBS containing 1% BSA after blocking with 10% human serum. Cells were analyzed on the ZE5 Cell Analyzer.

When CD3SBV440 was added to the panel from vendor two, Figure 4, CD3⁺CD4⁺ T cells could now be identified without affecting the other population identification. For all three panels tested, including StarBright dyes with minimal spillover as the additional markers, meant no compensation was required. The inclusion of more dyes may require compensation.

Fig 4. Adding SBV440 to a three-color cocktail from vendor two. Human peripheral blood was stained with A, the three-color premixed cocktail of CD4, CD127, and CD25, or B, the three-color cocktail plus PureBlu DAPI (1351303) and CD3SBV440 (MCA463SBV440). The gating strategy selected the live, single cells, then the lymphocytes. Shown are the CD3⁺ T cells, CD3⁺ and CD4 T helper, and the CD127^hi CD25^lo Treg cells.  All staining was performed at 4^oC in PBS containing 1% BSA after blocking with 10% human serum. Cells were analyzed on the ZE5 Cell Analyzer.

No Special Buffer Required

These data confirm that StarBright Dyes can be used in any common buffer and are flexible enough to be combined with any other fluorescent dyes, including those in premixed panels, without the need to change your staining protocol. Furthermore, as they do not require a special buffer, multiple StarBright Dyes can be used without changing your experiment.

Visit our dedicated StarBright Dye page to find out more about the dyes and to see which antibodies are available for you to use in your experiments.