HomeGuides, Tips, and TutorialsPanel Design: Dos and Don’ts

Panel Design: Dos and Don’ts

Things to Do:

  • Prepare, prepare, prepare.
  • Know your instrument configuration (determines fluorochromes and dyes you can use).
  • Understand fluorochrome brightness.
  • Know the antigen density as it pertains to your application (activation, etc).
  • Know your gating strategy ahead of time – what we are now referencing as a “Biological Tree” – to determine co-expression.
  • Pair antigen with fluorochrome to maximize resolution (Dim Ag on moderate to bright fluorochromes and bright Ag on moderate to dim fluorochrome). Be mindful of co-expressing markers.
  • If possible, fix your sample if you’re not analyzing soon after prep (fixatives need to be checked against fresh samples to see if possible.)
  • Know the fluorochrome/dye excitation and emission spectra (FITC, AF488, GFP, CFSE, SybrGreen…. Are all measured in the same channel)
  • To minimize compensation spillover try to choose the primary detector off each laser first then pay attention to similar spectra emissions.
  • Re-suspend cells in proper buffers.
  • Optimize staining by titrating the antibodies before use.
  • Check you are using the correct reagent species and clone.
  • Include viability dyes – Fixable viability stains for IHC or to analyze at a later time.
  • Be sure you have the appropriate controls: Cells only, single stained controls for spillover, biological/experimental controls, FMO’s.
  • Use appropriate blocking reagents to mitigate unspecific binding.
  • Be sure the instrument has been properly QC’ed before use.
  • Run samples on appropriate flow rate (ex – Cell Cycle should be run on low to minimize CV.)
  • Choose your PMT voltages optimized for the best resolution of signal.
  • Protect samples from light.
  • Keep samples at the appropriate temperature.
  • Understand you may need to change the panel once you see the first set of data.
  • Contact your flow core or BD support if you have questions sooner than later.

The Don’ts

  • Add more antibody to stain – more isn’t better.
  • Assume every flow experiment is the same as your lab member – IHC is different than surface labeling, which is different than cell cycle, etc.
  • Don’t measure the spillover for a reagent with a different one – ex: don’t use a AF488 single stained control for a FITC sample.
  • Don’t assign co-expressed markers in channels that have large spread.
  • Don’t take shortcuts.