Premixing StarBright Dyes

Problem

Preparing master mixes for large flow cytometry panels can be time consuming, and if multiple samples are to be tested over a long period time, there will be some differences in the master mixes resulting in errors. One way to avoid this is to prepare one master mix which can then be used over a long period of time.

However how can you be assured that the premix will be stable over time?

Solution

Bio-Rad recently launched StarBright Dyes. These exceptionally bright dyes bring a new level of flexibility to your flow experiments, by allowing the resolution of rare populations and low density antigens, without the requirement for special buffers even when multiplexing.

StarBright Dyes can be premixed, with no loss of performance for up to 28 days.

The data below demonstrate this.  

Proof

We premixed four StarBright Dyes, CD19SBV515, CD3SBV610, CD4SBB700, and CD8SBV670, and stored as a master-mix at 4oC. We then compared the staining of the freshly made master mix, a mix prepared 1 week before use, and a mix prepared 4 weeks before use on the same sample. As you can see in Figure 1, the pattern of staining is very similar regardless of the age of the master mix, with similar B and T cell populations identified.

Fig. 1. Staining of peripheral blood with premixed StarBright Dyes


Fig. 1. Staining of peripheral blood with premixed StarBright Dyes. Human peripheral blood was stained with a mix of CD3SBV610 (MCA463SBV610), CD4SBB700 (MCA1267SBB700), CD8SBV670 (MCA1226SBV670), and CD19SBV515 (MCA1940SBV515) made up fresh or made and left at 4oC for up to 28 days.

We also tested the effect of leaving the StarBright Dye mixes in different buffers. We compared using no buffer, which was just PBS, Bio-Rad Staining Buffer (BUF073) which contains FBS, 1% BSA in PBS, and Brilliant Stain Buffer (BD). As can be seen from the graphs (Figure 2), apart from some minor variation when no buffer was used, there was very little variation in the staining over time, regardless of the buffer. The staining was very consistent and the same percent positive for each population could be identified. Furthermore, as can be seen in the two-color plots showing all combinations (Figure 3), there were no unusual staining patterns or interactions regardless of the length of time the panel was left at 4oC for.

Fig. 2. Effect of different buffers on premixing.


Fig. 2. Effect of different buffers on premixing. Graphs of percent positive for the CD3+CD4+, CD3+CD8+, and CD19+ populations, when StarBright Dyes were stored in different buffers after premixing for up to 28 days.

Fig. 3. NxN plots of all StarBright Dyes in the panel.


Fig. 3. NxN plots of all StarBright Dyes in the panel. All of the two-color plot combinations for the StarBright premixed and freshly made up panels reveal identical staining, regardless of the length of time the premixed panels were stored for.

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