Flow Cytometry Compensation Beads

On this page:

• Compensation Bead Selection Guide
• OneComp and UltraComp eBeads
• Fluorescence compensation
• AbC Total Antibody and ArC Amine Reactive Compensation Beads
• GFP BrightComp eBeads

When using two or more fluorochromes in a single experiment, some portion of the emission spectra of a given fluorochrome may fall within the detector of another fluorochrome. To ensure that the fluorescent signal measured is due to the fluorochrome of interest, the process of compensation is used to mathematically subtract the signal from the fluorochrome that is spilling over into that channel. To calculate how much compensation is needed, single-color control samples must be run with each experiment. The signal from a single fluorochrome that is detected in each of the other channels can be measured and compensation applied to remove the signal. Beads, such as Invitrogen UltraComp eBeads or OneComp eBeads, AbC Antibody Compensation Beads, or cells stained with the antibodies used in the experiment can be used as single-color controls.

Figure 1. Compensation is the mathematical correction flow cytometry data to remove spectral overlap.

Compensation beads are designed to provide a consistent, accurate, and simple-to-use tool to set flow cytometry compensation. Consistently fluorescent compensation beads help users avoid sub-optimal compensation that can occur when dimly fluorescent sample cells are used to set compensation. Compensation beads exhibit superior utility in the compensation of multicolor experiments, and can also be used with the same antibody as was used in the experiment (instead of, for example, a bright CD4 substitute).

Compensation beads are suitable when:

• Strong signal for compensating a positive population is needed 
• Performing multiple plates or large experiments
• Cells are in limited supply for making compensation controls

OneComp eBeads are polystyrene microspheres that contain a mixture of antibody-coated, positive compensation beads and uncoated, negative compensation beads, combined in one vial. Dispense the eBeads as a single drop for compensation made easy. UltraComp eBeads microspheres were developed with the same benefits as OneComp eBeads microspheres with the added advantage of being optimized for use on the violet laser.

Learn more about OneComp and Ultracomp eBeads

Figure 2. Staining of UltraComp eBeads with 13 different Invitrogen eFluor 450 dye–conjugated monoclonal antibodies, including one of each subclass commonly used in flow cytometry. Beads were stained with 0.25 µg of each antibody and analyzed by flow cytometry. Each histogram represents one staining antibody (clone and isotype indicated at right, except for Rat IgG2a and Hamster antibodies for which the isotype and clone are indicated).

AbC Total Antibody Compensation Kit

The three AbC compensation bead kits contain two types of specially modified polystyrene microspheres: 1) AbC capture beads (also called positive beads), which bind all isotypes of the specific immunoglobulin, and 2) negative beads, which have no antibody binding capacity. After incubation with a fluorophore conjugated primary antibody (mouse, rat, hamster, or rabbit, depending on the kit used), the two components provide distinct positive and negative populations of beads that can be used to set compensation. The AbC bead kits were designed such that the negative beads are added after labeling of the positive bead in order to avoid any transfer of fluorescence over time to the negative bead.

ArC Amine Reactive Compensation Beads

The Invitrogen ArC Amine Reactive Compensation Beads were developed with the same laser compatibility and two-vial format as AbC Total Antibody Compensation Beads, but were specifically designed for compensation of the Invitrogen LIVE/DEAD Fixable Dead Cell Stains.

Figure 4. Staining profile of the ArC Amine Reactive Compensation Bead Kit components with 3 LIVE/DEAD Fixable Dead Cell Stain kits. (A) LIVE/DEAD Fixable Violet dye stained beads were analyzed with 405 nm excitation, emission was collected with a 450/50 nm bandpass filter. (B) LIVE/DEAD Fixable Green dye stained beads were analyzed with 488 nm excitation, emission was collected with a 525/50 nm bandpass filter. (C) LIVE/DEAD Fixable Far Red dye stained beads were analyzed using 633 nm excitation, emission was collected with a 660/20 nm bandpass filter.

The GFP BrightComp eBeads provide a consistent, accurate and simple-to-use reagent for setting flow cytometry compensation when using green fluorescent protein (GFP). The product includes modified microspheres to allow for easy compensation of samples with different levels of GFP expression (Figure 5). Each drop of beads contains negative beads and positive beads stained with a dye that has a near-identical, spectral match to GFP at 3 levels of intensity (Figure 6). Dispense the GFP BrightComp eBeads as a single drop for GFP compensation made easy using nearly any flow cytometer.