In a perfect world, the fluorescence emission profile for each individual fluorophore would be a very intense, narrow peak, well separated from all other emission peaks. In reality, organic dyes and fluorescent proteins have broad emission peaks. Compensation beads are used to ensure the integrity of your experimental data during or after data acquisition. Since cellular-based compensation controls are not always completely effective, using bead-based compensation controls is a good alternative. These flow cytometry compensation beads give you more consistent and reliable compensation because they have low lot-to-lot variability and have been optimized for the application you are running. Bead-based flow cytometry compensation controls also have a history of long-term stability due to their manufacturing processes.

Flow Cytometry Compensation Beads Selection Guide

Product name Assay type Positive bead Negative bead Cat. No.
AbC™ Total Antibody Compensation Bead Kit Immunophenotyping Hamster, mouse, rabbit, and rat antibodies* No binding capacity A10497
AbC Anti-Mouse Bead Kit Immunophenotyping Mouse monoclonal antibodies* No binding capacity A10344
AbC Anti-Rat/Hamster Bead Kit Immunophenotyping Rat and hamster monoclonal antibodies* No binding capacity A10389
ArC™ Amine Reactive Compensation Bead Kit Cell viability assay Amine reactive dyes and LIVE/DEAD™ Fixable Dead Cell Stains No amine reactive capacity A10346
*AbC capture beads bind all isotypes.

Compensation beads for Immunophenotyping

AbC Compensation Bead Kits

  • An alternative to using precious samples for setting flow cytometry compensation
  • Highest reactivity to different subclasses of mouse, rat, and hamster immunoglobulin
  • Fast and simple bead-based fl ow cytometry compensation
  • Removal of inconsistencies due to variations in antigen expression

AbC™ bead kits provide a consistent, accurate, and simple-to-use technique for the setting of flow cytometry compensation when using:

  1. Fluorophore-conjugated mouse, rat, hamster, or rabbit antibodies (AbC Total Antibody Compensation Bead Kit) (Figure 1)
  2. Fluorophore-conjugated mouse antibodies (AbC Anti-Mouse Bead Kit) or
  3. Fluorophore-conjugated rat or hamster antibodies (AbC Anti-Rat/Hamster Bead Kit)

All three 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. You can perform compensation with the same fluorophore labeled antibody used for cell staining. The consistent nature of bead scatter and high surface antibody-binding capacity allows you to more consistently and accurately set compensation for any combination of fluorophore-labeled primary antibodies. Both types of microspheres (the AbC™ capture beads and negative beads) have a diameter of approximately 6 μm (actual size for each lot is listed on the component vial). The bead suspensions are supplied in dropper vials for convenient sample application.

Four histograms showing staining of the AbC™ Total Antibody Compensation Bead Kit
Figure 4. 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.

Compensation beads for LIVE/DEAD Fixable Dead Cell Stains

ArC Amine Reactive Compensation Bead Kit

  • Eliminates the hassle of heat-treating cells
  • Optimized for all LIVE/DEAD Fixable Dead Cell Stain kits
  • Fast and simple bead-based flow cytometry compensation
  • An alternative to using precious samples for setting compensation
  • Accurate and consistent results

The ArC™ Amine Reactive Compensation Bead Kit provides a consistent, accurate, and simple-to-use technique for the setting of flow cytometry compensation when using any of the LIVE/DEAD Fixable Dead Cell Stain Kits or when using any amine reactive dye. LIVE/DEAD Fixable Dead Cell Stain Kits (and amine reactive dyes) can be used to evaluate mammalian cell viability based on the fact that the dye reacts with cellular amines. The reactive dye can enter the cell via compromised membranes of necrotic cells and react with free amines in the interior and on the surface of the cell, resulting in intense fluorescent staining. In contrast, only the cell-surface amines of viable cells are available to react with the dye, resulting in relatively dim staining. The difference in fluorescence intensity between the live and dead cell populations is typically greater than 50-fold.

The ArC Amine Reactive Compensation Bead Kit includes two types of specially modified polystyrene microspheres to allow easy compensation of the LIVE/DEAD Fixable Dead Cell Stains: the ArC reactive beads (Component A), which bind any of the amine reactive dyes, and the ArC negative beads Component B), which have no reactivity. After incubation with any amine reactive dye, the two kit components provide distinct positive and negative populations of beads that can be used to set compensation (Figure 2). The ArC Amine Reactive Compensation Bead Kit can be combined with the AbC Total Antibody Compensation Bead Kit for use with fluorophore conjugated mouse antibodies, allowing even more consistent and accurate compensation for multicolor immunophenotyping experiments that also incorporate a LIVE/DEAD Fixable Dead Cell Stain.

three-panel histograms showing the staining profile of the ArC Amine Reactive Compensation Bead Kit components with three LIVE/DEAD Fixable Dead Cell Stain Kits

Figure 2. Staining profile of the ArC™ Amine Reactive Compensation Bead Kit components with three 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.