Flow Cytometry Panel Builder

Multicolor flow cytometry with 5 easy steps

With this tool, you can:

Create a new immunophenotyping experiment or add antibodies and reagents to an older panel

Insert antibodies already used in your experiment

View fluorochrome spectrums to quantify emission spectral overlap

Export an excel document with your antibody choices or directly order

Getting started with the flow cytometry panel builder tool

Choosing the optimal combination of fluorochromes can be simplified with a guided method. The Invitrogen Flow Panel Builder offers a customizable panel building process to fit your flow cytometry experimental needs, whatever your experience level.

Video: how to use the panel builder

Watch the video to learn how to use the Invitrogen Flow Cytometry Panel Builder to build your next flow cytometry panel in 5 easy steps.

Common fluorochrome brightness

Figure 2. Average stain index of fluorochrome conjugated to CD4 and analyzed on the Attune NxT Flow Cytometer.

Fluorophores emit light with varying levels of brightness (Figure 2). When choosing fluorochromes on the panel builder, we suggest:

  • Have a staining index for your specific flow cytometer. A staining index can be provided as a chart or table from a manufacturer who tested the same clone with different conjugates on their instrument. It is also possible to create a staining index with flow capture beads. For example, Figure 2 shows a staining index of the most popular fluorochromes analyzed on the Attune NxT Flow Cytometer. Each flow cytometer has different lasers, filters, and detectors that can influence the signal observed from a marker conjugated to a fluorochrome.
  • When designing a multicolor experiment, choosing only very bright fluorochromes may result in spillover and can dampen the signal of a marker.
  • Examine the mean fluorescence intensity for each fluorochrome and antibody clone to compare brightness. These can be found in product data sheets.

Want to know more about a staining index? Need the staining index for the BD™ LSR II Flow Cytometer? Find it in our Flow Cytometry Panel Design: Basics

Strategies for identifying immune cell markers

Each channel on your flow cytometer can report on the presence of a marker when it is bound by a fluorochrome-conjugated detection molecule. Many publications give lists of markers for various detection strategies, but in general we recommend:

  • Employing a strategy to detect a combination of positive, negative, and functional markers to identify a cell. More markers give a more comprehensive analysis of a cell population and provide more confidence in a cell identification.
  • Accounting for gene and protein expression changes when cells are stimulated or isolated from different sources.
  • Using Optimized Multicolor Immunofluorescence Panels (OMIPs) to aid in the design or expand a panel. Each publication provides (1) information about the cell populations, antigen density, and marker co-expression; (2) optical configuration of the instrument, including excitation wavelengths and emission filters available; (3) antibody characteristics; (4) fluorophore characteristics.

Looking for markers to detect your cells? We have a guide for human and mouse cellular markers.

Natural Killer cells
Tregs
B cell
T cell

Figure 3. Immune cells may be identified by viability dyes in combination with labeled antibodies targeted to markers that are specific to each cell.

Relative antigen density for common immune cell markers

Choosing an antibody should be based on both antigen abundance and fluorochrome brightness to minimize compensation and help ensure good resolution of cell populations. We advise:

  • Using antigen density tables as a guide (see below for examples of antigen density for popular T and B cell markers). Gene expression levels may vary depending on cell source, stimulation, and protocols used for fixation.
  • Dim fluorochromes to detect highly expressed proteins and bright fluorochromes to detect less-abundant proteins.
  • Use flow cytometry compensation beads to help determine the specific density of your antigen. Internet-accessible databases can also help to find RNA expression levels.
Table 1. Antigen density for popular T cell markers.
Targets Typical expression levels
CD3 High
CD4 High
CD8 High
CCR7 Low to medium
IL-2 Medium
IFN gamma Medium to high
Phospho-CD247 (CD3z) Y142 Medium to high
CD45RA High
CD45RO Medium to high
CD27 Medium to high
CD28 Medium to high
CD122 Medium to high
CCR4 Low to medium
CD127 Low to medium
CD25 Low to medium
CD132 Low to medium
Table 2. Antigen density for popular B cell markers.
Targets Typical expression levels
CD19 Low to medium
IgM Low
CD20 High
CD34 High
CD25 High to medium
CD27 Low
CD138 Low
CXCR4 High to medium
IL-10 High to medium

Quick tips and tricks for your best panel

R&D scientist Natalie Oxford share her learnings for a flow cytometry panel that can help you get published.

Tip 1: Minimize spill-over by using the correct and separate channel

Tip 4: Save your bright fluorochromes for dim targets

Tip 2: Intracellular targets need special buffer for fixation and permeabilization for staining

Tip 5: Try to combine negative markers in one channel (dump channel) to save space on your panel

Tip 3: Viability dyes are required to find live cells