Take the guesswork out of compensation

Compensation beads are recommended when performing flow cytometry using multiple channels, markers that are poorly expressed, or when sample is limited. Invitrogen™ OneComp eBeads™ compensation beads and the Invitrogen™ AbC Total Antibody Compensation Bead kit are designed to give you the options you need for flow cytometry compensation. 

Fluorescence compensation

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.

Spectral overlap illustration

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

 

Advantages of compensation beads

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 and UltraComp eBeads

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

UltraComp eBeads staining chart

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 and ArC Amine-Reactive Compensation Beads

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.

Four histograms showing staining of the AbC™ Total Antibody Compensation Bead Kit

 

Figure 3. Histograms showing staining of the AbC Total Antibody Compensation Bead Kit. The histograms show the signal separation of the positive capture beads from the negative beads after binding to mouse (top left), rat (top right), and hamster (bottom left) monoclonal antibodies, and rabbit (bottom right) mono- and polyclonal antibodies. Capture beads were labeled with an optimized amount of each PE antibody conjugate and analyzed on an Attune™ Acoustic Focusing Cytometer using 488 nm excitation and a 574/26 nm bandpass filter.

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.

ArC compensation bead kit histograms

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.

 

Compensation beads selection guide
  UltraComp eBeads OneComp eBeads
Reactivity Hamster, mouse and rat antibodies and recognition of the kappa & lambda chains
Format One vial, dispense as a single drop
Laser compatibility Compatible with most standard lasers, UV to 633 Not compatible with violet lasers
Size 25 tests 100 tests 25 tests 100 tests
Cat. No. 01-2222-41 01-2222-42 01-1111-41 01-1111-42
  AbC Total Antibody Compensation Bead Kit AbC Anti-Mouse Compensation Bead Kit AbC Anti-Rat/Hamster Compensation Bead Kit
Reactivity Hamster, mouse, rabbit and rat antibodies* Mouse monoclonal antibodies* Rat and hamster monoclonal antibodies*
Format 1 vial positive bead;
1 vial negative bead
1 vial positive bead;
1 vial negative bead
1 vial positive bead;
1 vial negative bead
Laser compatibility Compatibility with most standard lasers, UV to 633 nm
Size 25 tests 100 tests 100 tests 100 tests
Cat. No. A10513 A10497 A10344 A10389
*AbC capture beads bind all isotypes.
  ArC Amine Reactive Compensation Bead Kit
Reactivity LIVE/DEAD fixable dead cell stains and similar amine reactive dyes
Format 1 vial positive bead; 1 vial negative bead
Laser compatibility Compatible with most standard lasers, UV to 633 nm
Size 25 tests 100 tests
Cat. No. A10628 A10346