Panel Design

When designing a panel, it is important to develop a strategy that provides maximum signal detection with minimum background in the channels for protein targets that are difficult to detect.

Signal overlap is a common form of background in which signal derived from a probe is measured in undesired channels. One of the chief advantages of mass cytometry is that signal overlap is small and measureable.

Panel design for cytometry experiments is the art of pairing probes (generally antibodies) with detected-molecules (fluorophores for flow or conjugated lanthanides and cadmiums for mass cytometry) so that expression differences are maximised and emphasised.

The Maxpar® Panel Designer (Fluidigm) is an interactive web-based application that simplifies and optimizes panel design. It allows users to build a panel consisting of MCAs from the Fluidigm® catalog and from custom conjugates.

The tool calculates and provides visualization of predicted signal overlap for each panel, and also has an algorithm that optimizes the metal tag selections for each target to optimize panel performance. Panels can be saved and exported for sharing with collaborators and for future import into CyTOF® instrument control software.

You can find the Fluidigm MaxPar Panel Designer User Guide here: Link (SharePoint)

To start designing your panel,
you need to create a login at
https://www.fluidigm.com/area-of-interest/panel-design or https://pdv2.dvssciences.com/login

The 2 basic rules of Panel design

DO:         Measure the most important markers in the most sensitive channels

Do NOT:   Place markers that appear on the same cells in neighbouring (or interacting) channels

Guidelines for Panel Design

From the MaxPar Panel Desinger User Guide:

1. Low-abundance antigens:
   1. Choose high sensitivity channels.The CyTOF is most sensitive to metal tags in the 153-176 Da range
   2. Choose mass tags in channels which receive little or no crosstalk from other metals in the panel
   3. Consider amplifying low abundance signal:
      • Use metal-conjugated anti-biotin or anti-FITC secondary antibodies
      • Stain a low abundance antigen simultaneously with more than one metalconjugated antibody, each directed against a different epitope, all conjugated to the same isotope.
2. High-abundance antigens:
   1. Choose mass tags which contribute little or no crosstalk signals to the mass channels used for low-abundance antigens
   2. Do not place a high abundance antigen in a channel that is -16 Da or +/-1 Da relative to the channel of a low abundance antigen
   3. Titrate antibody and use at the lowest concentration possible so as to minimize crosstalk impact to other mass channels.
3. Antigens with variable expression:
   1. Treat as low abundance and follow guidelines for mass tag choice as outlined above.
   2. Perform pilot experiments to determine the biological dynamic range of abundance levels.
   3. Titrate staining reagents such that condition of lowest abundance is detectable.
4. Identify targets that are mutually exclusive of one another (CD4 and CD20, for example). It is acceptable if two such antibodies are bound to metals that crosstalk if the identified populations can be gated independently.

For the Helios, the area of the mass range that is most sensitive is around 153-165. These channels should be used for our most important or lowest-expressed antigens of interest . Furthermore, there are also differences at either end of the mass range.

Around 140 is the area of least sensitive detection for lanthanides. The area around 170+ is lower sensitivity than around 160 but better than at 140. The masses of the cadmium isotopes are 106-116.