Panel Design
Multicolor flow cytometry is a powerful technology that enables simultaneous analysis of multiple markers at the single-cell level. With the increase in number of 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 set-up
- Explore pre-designed panels at the Interactive Cell Map
Step 1
Define your experimental hypothesis
Defining your experimental hypothesis is the first step in panel design. Start with identifying:
- The biological information you are trying to achieve
- The population(s) of cells you wish to interrogate
- Whether targets are found on the cell surface or intracellularly
Step 2
Marker selection
During the second step of the panel design process, you will need to identify which and how many markers you need to identify the population of interest.
Pay attention to:
- Marker expression levels
- Primary antigen: Expressed at high density, often defining lineages
- Secondary antigen: Often expressed over a continuum
- Tertiary antigen: Critical markers expressed at low density
- Marker coexpression, especially of dim markers
- The gating strategy needed to identify the population(s) of cells you wish to interrogate
Step 3
Know your flow cytometer
Knowing your instrument is essential. Understanding your instrument's configuration will let you know how many markers and which fluorochromes your instrument can detect.
Elements to consider include:
- Laser wavelength for excitation
- Number of detectors for each laser
- Filters available to detect the fluorochromes
Step 4
Fluorochrome assignment
Carefully select fluorochromes to resolve markers at all expression levels and minimize spectral overlap. Consider using tools like a fluorochrome resolution ranking and a spectrum viewer to help assess:
- Fluorochrome resolution
- Cross laser excitation
- Fluorochrome spillover
Remember to pair bright fluorochromes with low expressing antigens and dim fluorochromes with high expressors. Keep in mind that spread only impacts the resolution of coexpressed markers.
Step 5
Review panel
Review your panel design and begin ordering your reagents. Remember to titrate your mass size reagents and optimize your staining protocol. Include proper controls for compensation, FMO and biological controls to help ensure optimal panel performance.
To find more panel design tips and some fun facts, download the “Flow cytometry panel design journey” infographic.
In addition, we also provide several tools, such as Spectrum Viewer, absorption and emission spectra, and buffer compatibility tool.
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
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