Flow Cytometry Panel Design Journey

Panel design is one of the most crucial parts of a flow cytometry experiment. It involves selecting the right combination of marker and fluorochrome for a given population of cells. But a badly designed panel can mean overlapping emission spectra, leading to the lost resolution of cell populations. Your choice of fluorochrome depends on a variety of factors, including antigen expression level, emission and absorption wavelengths, laser and instrument settings, and the biology of the experiment.

It is very important for anyone starting to explore flow cytometry to understand the principles of panel design and correctly implement them. While a lot of this will come from trial and error, having a working knowledge of these principles, plus lasers, fluorochromes, and panel design can help simplify the learning curve considerably. The best way to acquire such knowledge is to listen to experts in the field and learn from their experience.

Multicolor flow cytometry is a powerful way to enable the simultaneous analysis of multiple markers at the single-cell level. With an increase in detectable parameters, the design of a multicolor panel can be challenging and requires an understanding of several factors that can influence panel performance:

  • Biology: Antigen density and co-expression
  • Fluorochrome: Brightness and spillover
  • Instrument: Configuration and setup

Click here (PDF, 12.7 MB) to explore pre-designed panels.

Step 1: Define Your Experimental Hypothesis

This is the first step in panel design (PDF, 2.6 MB). Start by identifying:

  • The biological information you want to gather
  • The population(s) of cells you wish to interrogate
  • Whether targets are found on the cell surface or intracellularly
Define Your Experimental Hypothesis

Step 2: Marker Selection

During the second step of the panel design process, you will need to determine which and how many markers you will need to identify the population of interest.

Pay attention to:

  • Marker expression levels
    • Primary antigen: Expressed at a high density, often defining lineages
    • Secondary antigen: Often expressed over a continuum
    • Tertiary antigen: Critical markers expressed at a low density
  • Marker co-expression, especially dim markers
  • The gating strategy needed to identify the population(s) of cells you plan to interrogate

Step 3: Know Your Flow Cytometer

Knowing your instrument is essential and understanding your instrument's configuration will help you determine how many markers and which fluorochromes it can detect.

You should consider the:

  • Laser wavelength for excitation
  • Number of detectors for each laser
  • Filters available to detect the fluorochromes
Know Your Flow Cytometer

Step 4: Fluorochrome Assignment

Carefully select fluorochromes to resolve markers at all expression levels and minimize spectral overlap. Consider tools like fluorochrome resolution ranking and spectrum viewers to help assess:

  • Fluorochrome resolution
  • Cross laser excitation
  • Fluorochrome spillover

Remember to pair bright fluorochromes with low-expressing antigens and dim fluorochromes with high-expressing antigens. Keep in mind that spread only impacts the resolution of co-expressed markers.

Step 5: Review Panel

Review your panel design and begin ordering your reagents. Remember to titrate your reagents and optimize your staining protocol. Include proper controls for compensation and FMO, and biological controls to help ensure optimal panel performance.

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Flow Cytometry Panel Design Journey

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BD Biosciences