Premixing StarBright Dyes

Following best practices, a small panel of four StarBright Dye conjugated antibodies, CD3 SBV610 (MCA463SBV610), CD4 SBB700 (MCA1267SBB700), CD8 SBV670 (MCA1226SBV670), and CD19 SBV515 (MCA1940SBV515), was designed. Antibodies were titrated before use and used in the panel at a concentration that gave the best stain index. We premixed these four StarBright Dyes and stored them as a master mix at 4^oC for 28 days. We compared the staining of the freshly made master mix and the mix prepared 28 days before use on the same sample of peripheral blood. 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 human blood with a premixed four StarBright Dye panel. Human peripheral blood was stained with an antibody mix consisting of CD3 SBV610 (MCA463SBV610), CD4 SBB700 (MCA1267SBB700), CD8 SBV670 (MCA1226SBV670), and CD19 SBV515 (MCA1940SBV515) made up fresh, or made with no additional storage buffer added, and kept at 4^oC for 28 days. DRAQ7 was added just prior to acquiring on the ZE5 Cell Analyzer and cells were gated on live single cell lymphocytes.

We also tested the effect of leaving the StarBright Dye mixes in three different buffers. We compared the samples stored in PBS (no buffer), Bio-Rad Staining Buffer (BUF073) which contains FBS, 1% BSA in PBS, and Brilliant Stain Buffer (BD), taking the staining volume to 50 µl/test. As can be seen from the graphs (Figure 2), apart from some minor variation when no buffer was used, there was very little difference 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.
 

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

Once we had established the buffer had no effect on the premixed vs fresh staining profiles, we expanded the panel size and incubated the mix for 33 days at 4^oC. By increasing the number of markers, we were able to detect more cell types. The new panel consisted of five StarBright UltraViolet and six StarBright Violet Dyes as shown in Table 1. Best practice in panel building was followed by titrating all antibodies prior to use, placing bright dyes on lower antigen density markers, and reducing the effect of spillover by placing those with high spillover on mutually exclusive markers.

Table 1. Antibodies used in premix staining panel to identify cell subsets in human peripheral blood.

As can be seen in Figure 3, the pattern of staining is very similar between the two master mixes, with similar T cell, B cell, monocyte, and granulocyte populations identified. Furthermore, as can be seen in the NxN two-color dot plots showing the staining of all lymphocyte markers (Figure 4) there was identical staining, with no interactions between the StarBright Dyes regardless of whether the panel was made fresh or incubated at 4^oC for 33 days.
 

Fig. 3. Staining of human blood with a premixed StarBright Dye panel. Human peripheral blood was stained with an antibody mix consisting of StarBright UltraViolet and StarBright Violet Dyes with no additional storage buffer. DRAQ7 was added just prior to acquiring on the ZE5 Cell Analyzer. Cells were gated on live single cells and plots shown after staining with an antibody master mix prepared fresh or kept at 4^oC for 33 days.
 

Fig 4. NxN plots of StarBright Dyes staining lymphocytes in the panel. NxN plots showing all of the two-color dot plot combinations for all StarBright Dye conjugated antibody staining of live single cell lymphocytes for the freshly stained and 33 day premixed panel.

We have demonstrated that premixing StarBright Dye conjugated antibodies for use in conventional cytometry on the ZE5 Cell Analyzer gives stable, reproducible data. Full spectrum cytometers provide a more stringent test of fluorophore stability. High data quality from spectral flow cytometers is reliant on accurate unmixing, minor differences in any part of the spectra of the fluorophores used in the full panel and single stained controls will give unmixing errors leading to false positives and unreliable data.

Here we show that StarBright Dyes can be stored in a premixed panel at 4^oC for up to 3 months with great results. The panel contains nine antibodies conjugated to StarBright Dyes, as shown in Table 2. All antibodies were titrated prior to use in the panel.

Table 2. Antibodies used in the three month premixed staining panel to identify major cell subsets in human peripheral blood.

The premix was tested at three months on a 5-laser Cytek® Aurora. The staining pattern on human peripheral blood was compared to a freshly made master mix. The two panels showed little variation in staining, as shown in Figure 5.

Fig. 5. Immunophenotyping data from human peripheral blood stained with a three month panel on a spectral flow cytometer. Human blood was stained with a multiplexing panel. A. freshly prepared, or B. stored at 4^oC for 3 months. Cells were stained with a live/dead dye, VivaFix 649/660, prior to antibody staining. Data was acquired on a 5 laser Cytek Aurora. Cells were gated on live, single cell lymphocytes and monocytes, not shown. 

For some experiments, there may be a need to store the premix for a longer period. An example is in longitudinal studies when the same panel is used to stain multiple samples obtained over an extended period.

Larger panels may also be required, panels can be expanded by the addition of more StarBright Dyes or different fluorophores, such as the more traditional ones; PE and FITC. However, not all fluorophores are suitable for premixing, for example they may require a special buffer. These fluorophores can still be included in the panel but added at the point of staining rather than in the premixed cocktail. The time taken to assemble large panels and reproducibility are still improved even when not all antibodies can be premixed.

Here we show that StarBright Dyes can be stored in a premix panel along with antibodies conjugated to FITC, PE and Alexa Fluor 700 for up to 12 months at 4^oC. The panel used is shown in Table 3. All antibodies were titrated prior to use in the panel.

Table 3. Antibodies used in the 12 month premix staining panel conjugated to StarBright Dyes or conventional fluorophores to identify cell subsets in human peripheral blood.

The premix was tested at regular intervals over the 12 month period by comparing the staining pattern on human peripheral blood with a freshly made master mix. Even when stored for up to 12 months the premixed panel showed no dye interactions and plots showed little variation compared to a freshly made panel, as shown in the data from the 12 month time point in Figure 6.

Fig. 6. Staining of human blood with a fresh and 12 month premixed panel. Human peripheral blood was stained with antibodies conjugated to StarBright Dyes, FITC, PE or A700, A. freshly prepared, or B. stored at 4^oC for 12 months. DAPI was added just prior to acquiring on the ZE5 Cell Analyzer. Cells were gated on live, single cells, not shown.

It is advisable to validate panels prior to use as a premix. Not all fluorophores are suitable to be included in premixed panels due to potential changes in spectral profiles, for example tandem dyes, or interactions between fluorophores that can be present with some polymer dye combinations. Also be aware that as more different types of fluorophores are included there may be changes in the concentration of buffer components used to maintain stability. This could alter the stability and the integrity of the pre-mixed panel may not be maintained.