Pushing the Limits

Ways of Expanding Immunophenotyping Panels

Advances in cell biology have expanded applications of immunophenotyping in drug discovery, diagnosis, and treatment planning. This has led scientists to design flow cytometry experiments with bigger panels, allowing an ever-increasing complexity of biomarkers. Expanding panels is tricky as it increases the complexity of design, workflow, and data analysis. Creating a panel is often compared to solving sudoku puzzles as there are many variables to be considered.

One way to expand your panel is to increase the number of lasers and detectors. By doing so, more fluorophores can be used in the panel. This requires instrument upgrades and increases costs. But what if you are already at the maximum possible limit of lasers and detectors and still want to increase panel size? Well, another option is to move from conventional to full spectrum flow cytometry. Thus, you may require instrument upgrade or an entirely new flow cytometer with spectral capabilities. This adds extra cost and significant resources for setup, training etc., so it may not be always practical.

Can Flow Cytometry Panel Expansion be Simple?

As the saying goes “necessity is the mother of invention”. To accommodate the panel expansion needs, scientists can add UV and Violet lasers and compatible conjugated antibodies to their set-up or go the spectral analysis route. Brilliant Ultra Violet™ (BUV) and Brilliant Violet™ (BV) are brighter antibodies which can stain dim markers on cells and provide minimum spillover and maximum compatibility with UV and Violet lasers and detector channels. Thus, occupying UV and Violet detector channels with antibodies labeled with BUVs and BVs will reduce the compensation issues. Polymer based technology of BUVs and BVs make it ideal for both ‘traditional’ and ‘spectral’ flow cytometry.

Looking outward to acquire more lasers or making major hardware upgrades to your flow cytometer is possible and a great tool. However, this may not always be an optimal or a feasible solution. But what if there was a way to get more out of your flow cytometer (with unchanged instrument hardware)? What if you could expand your panel and achieve your panel design goals by making a slight change in the panel design and fluorochrome selection? Sounds ideal, right? Good news, it is achievable though selection of appropriate dyes and conjugates.

Ability to Control Conjugate Brightness Makes Panel Expansion Easier

Even with violet and UV in the mix, often there are situations where overlaps in the signal exist using common dyes. A novel solution is available for this problem as well. You don’t need to look any further than Invitrogen^TM NovaFluor^TM Dyes made with Phiton technology (macrostructure labeled with small-molecule fluorophores). NovaFluors unlocks previously unusedable channels in current instrumentation. They also provide high resolution data for cleaner single-cell analysis. These dyes can thus serve as a ‘gap filler’ along with BUVs and other dyes. Along with variable brightness for NovaFluor Blue 610 and NovaFluor Blue 660, NovaFluors have minimal cross-laser excitation, allowing the replacement of PE-tandem, dyes. Yes, you read that right! Now you can increase your conventional panel size without a complete panel redesign.

Novafluor dyes are also more stable than other tandem dyes, which increases their shelf life and drastically reduces lot-to-lot variability. In addition to BUVs and NovaFluors, SuperBright Polymer Dyes and eFluor Organic Dyes have unique spectral signatures and are compatible with both ‘traditional’ and ‘spectral’ workflows. Thus, providing you all the flavors needed for your perfect panel design.

Take Home (or to the Lab) Lesson

Using specialized dyes such as BUVs and NovaFluors in combination can bring versatility to ‘traditional’ and ‘spectral’ flow cytometry studies. You can spread your panels across a broader range with these dyes without significant changes to your panel design or instrument hardware. In short, BUVs and NovaFluors together can help you uncover the hidden potential of your flow cytometer.

Learn more about the product in this blog: Brilliant Ultra Violet™ (BUVs) and Brilliant Violet™ (BVs), Invitrogen^TM NovaFluor^TM Dyes

Resources and Solutions for Flow Cytometry

• Flow Cytometry Panel Builder – this online tool helps you design an effective reagent panel for your specific application on your conventional or spectral flow cytometer
• Flow Cytometry Protocols Handbook – protocols that fit your needs in flow cytometry ranging from sample preparation to numerous cell stimulation conditions, staining, immunophenotyping and data analysis strategies
• Immunology at Work Resource Center – practical guidance and protocols to help you get started quickly and confidently

Brilliant Violet™ and Brilliant Ultra Violet™ are trademarks of Becton, Dickinson and Company or its affiliates, and are used under license.