Five Steps to Successful Flow Cytometry Panel Design

Multicolor flow cytometry allows researchers to identify and characterize distinct cellular subpopulations within a heterogeneous sample. It achieves this using combinations of fluorochrome-labeled antibodies for detecting cell surface markers and other biomolecules, making panel design critical to its success. This article provides guidance for building robust flow cytometry panels and shares tips to help maximize the resolution of your population of interest.

Step 1: Define your experimental hypothesis

Panel design begins with defining your experimental hypothesis. This should include establishing the type of biological information you are trying to attain, and the populations of cells you wish to interrogate, as well as determining where, when, and how targets of interest are expressed. Interrogation of certain cellular targets may require some form of activation, while detecting both cell surface and intracellular markers often necessitates staining for extracellular targets first (prior to fixation, permeabilization, and intracellular staining) to avoid damaging cell surface epitopes. If secreted molecules such as cytokines are of interest, a protein transport inhibitor such as Monensin or Brefeldin A may be useful to trap the targets inside the cells.

Step 2: Select your markers

A key consideration for marker selection is the number of markers required to accurately identify the population of interest. Although certain subsets of cells can be identified using just one or two markers, it is more common for multiple markers to be used in combination. Marker expression levels are also important because marker density impacts panel design:

• Primary antigens are often well-characterized and typically exhibit expression that is either on or off; they are commonly used for defining cell lineages and include CD3, CD4, and CD19
• Secondary antigens are also usually well-characterized but are more often expressed over a continuum; they enable deeper interrogation of cellular subtypes and include CD27, CD28, and CD45RA
• Tertiary antigens are expressed at low density and are critical for cellular identification; examples include CD25, STAT5, and FoxP3, in addition to uncharacterized antigens

Assigning markers to each of these categories can help determine an appropriate strategy for cellular identification by ensuring the most difficult to detect antigens—which usually have the fewest commercially available antibody conjugates—are addressed first. Ultimately the markers selected should be compatible with a gating strategy that identifies the populations of cells to be interrogated.

Step 3: Familiarize yourself with your flow cytometer

Understanding the configuration of your flow cytometer is essential to determine how many markers can be measured simultaneously and which fluorochromes it is possible to detect. Factors to consider include the laser wavelengths used for excitation, the number of detectors off each given laser, and the filters available for fluorochrome detection. Fluorochrome excitation and emission maxima should be matched to the flow cytometer’s lasers and detectors, respectively; this process can be simplified by using online panel builder tools that match fluorochromes with a particular instrument.

Step 4: Assign your fluorochromes

Fluorochrome selection should aim to resolve markers at all expression levels and minimize spectral overlap. It is recommended that bright fluorochromes be paired with low expressing antigens, and vice versa, and that researchers consider using tools such as a fluorochrome resolution ranking or spectra viewer to assess factors such as cross laser excitation and the potential for spillover. Ideally, fluorochromes should be spread across as many lasers and detectors as possible to minimize the risk of unwanted background signal. Where the number of available lasers is a limiting factor, tandem dyes (two covalently linked dyes with optimized excitation/emission properties) can increase flexibility in panel design. It is also worth keeping in mind that spread only impacts the resolution of co-expressed markers; spillover between the emission spectra of mutually exclusive antigens will not be an issue since these will not be expressed together in the same cell gate.

Step 5: Review your panel

Once panel design is complete, it is important to perform a thorough review before ordering in the necessary reagents. Next, the staining protocol should be carefully optimized to avoid wasted time, resource, and precious material when the panel is used for sample testing. Critical factors for optimization include the concentration of any antibody reagents, the selection of a suitable viability stain, and the set-up of appropriate controls. The latter should encompass biological controls that comprise known positive and known negative sample types as well as treated and untreated samples where relevant; fluorescence minus one (FMO) controls for evaluating fluorescence spread; and compensation controls that address fluorochrome spillover and ensure only true staining is revealed.

BD Biosciences has developed an extensive portfolio of reagents, instrumentation, and software for flow cytometry, including easy-to-use resources that simplify panel design. For more panel design tips and tricks from BD, visit their Panel Design Resources page.