Strategies and Considerations for the Characterization and Deeper Immunophenotyping of T Cells
T cells develop distinct surface markers and cytokine signatures during their development. Different types of T cell subsets have been identified based on their phenotypic and functional characteristics. Carefully designed flow cytometry panels with the combination of some basic surface markers can be used for characterizing T cells at a high level. But for deeper characterization of the different subtypes, a basic panel is not sufficient. Development of larger multicolor panels with additional drop-in fluorochromes is needed. Expansion of an existing flow cytometry panel by adding new fluorochromes can be challenging as the drop-ins can introduce spillover. The spillover spread may compromise panel resolution, ultimately requiring a complete redesign of a new panel, influencing cost, time and the ability to define cell biology in depth.
How are standalone panels and pre-optimized backbone panels different?
Several standalone panels are available for T cell characterization. However, these panels are not specifically designed to accommodate drop-ins. Expansion of these panels can cause suboptimal resolution of backbone and/or drop-in channels if panel design rules are not strictly followed. In addition, the standalone panels incorporate some of the most commonly available fluorochromes instead of leaving them open for drop-in flexibility.
A strategically designed pre-optimized backbone panel eliminates these disadvantages. Both standalone and backbone panels use different approaches, but while both rely on the fundamental rules of panel design, such as considerations to fluorochrome brightness and spillover and antigen density and co-expression, they differ in several practical aspects:
| Standalone panel | Backbone panel |
|---|---|
| Designed for use without modification or additions | Prospectively designed to be expanded with different drop-ins |
| Easy to design—utilize spectrally separated fluorochromes | Challenging to design—utilizes challenging fluorochromes with high spillover |
| Difficult to expand—challenging selection of remaining fluorochromes | Easy to expand—spectrally separated fluorochromes are left for drop-ins |
| Panel redesign may be required to add new markers | Minimal panel design is required to add new markers in the backbone |
Considerations for building backbone panels
When building a backbone panel, the following four important criteria should be considered:
- — Backbone panel should clearly resolve all relevant cell subsets
- — Panel should not impact resolution of the recommended drop-in fluorochromes
- — Drop-in fluorochromes should not impact resolution of the backbone panel-Drop-in fluorochromes should not impact each other
The newly developed BD Horizon™ Human T Cell Backbone Panel from BD Biosciences
BD Biosciences has developed the BD Horizon™ Human T Cell Backbone Panel, which contains five individual vials of fluorochrome-conjugated antibodies against core T cell markers—CD3, CD4, CD8, CD45RA, CD197 (CCR7)—conventionally used to assess T cell maturation and identify naïve, central memory (CM), effector memory (EM) and effector memory RA (EMRA) subsets.1,2 The kit also contains the BD Horizon™ Brilliant Stain Buffer for optimal performance.
The BD Horizon™ Human T Cell Backbone Panel could be used as a starting point for a deeper dive into, for example, CD4⁺ T helper cells and further identification of regulatory T cells through the addition of specific drop-ins.
Advantages of using the pre-optimized BD Horizon™ Human T Cell Backbone Panel
The BD Horizon™ Human T Cell Backbone Panel is strategically designed to be complemented with up to five recommended drop-in fluorochromes paired with your antigens of choice, depending on instrument configuration, with minimal panel design effort and loss of resolution.
It is vigorously tested and provides application data that can be used to gauge the performance of the panel before using it. By reducing panel design effort and providing tested and pre-optimized panels, it helps in building efficiency and confidence in your experiments.
- — Strategic design of a backbone panel allows for expansion with recommended fluorochrome drop-ins, without impacting resolution
- — The panel design process for addition of new markers is simplified, as spillover spread and antigen co-expression are not of concern
- — Proper matching of fluorochrome brightness and antigen density is still required to ensure optimal resolution
- — The number of recommended drop-in fluorochromes may change depending on flow cytometer configuration
- — Application data demonstrate the flexibility and clear resolution of a properly designed backbone panel
If you are interested in learning more about the T cell backbone panel, reviewing performance data, accessing relevant protocols and getting additional tips and tricks, download our BD Horizon™ Human T Cell Backbone Panel e-book.
