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Compensation beads capture species-specific antibodies conjugated to fluorophores and other types of reagents. The purpose of these beads is to set voltages and gating parameters for obtaining accurate fluorescence signal.
Flow cytometry multicolor experiments may need compensation when there is fluorescence spillover (Figure 1). Pairing fluorochromes based on antigen density, fluorochrome brightness, and separating by channels helps minimize the effects from spillover and may remove the need for compensation from smaller experiments. However, as the number of parameters and colors increase, so does the complexity of removing overlapping signal. Calculating compensation requires controls including unstained, fluorescence minus one (FMOs), and single-color samples. Learn more about basics of panel design and flow cytometry controls.
Beads are recommended when:
Figure 1. An example of overlapping emissions from three fluorophores on the Invitrogen Flow Cytometry Panel Builder. Fluorescence signal may overlap if emission spectrums are broad and captured in a different detector specific for another fluorochrome. The overlap or spillover of this emission signal can provide false results. To remove fluorescence spillover, the mathematical process of compensation provides the signal of interest by subtracting the overlap between the two fluorochrome in the same channel.
Consideration needs to be taken when picking a compensation bead to set positive and negative signals for antibodies in a flow cytometry experiment. We offer several compensation beads specifically designed for flow cytometry antibodies, fluorescent proteins, and reagents.
UltraComp eBeads Plus compensation beads* | UltraComp eBeads compensation beads | AbC Total Antibody Compensation Bead kit* | OneComp eBeads compensation beads | |
---|---|---|---|---|
Application | Immunophenotyping | |||
Reactivity | Human, rabbit, hamster, mouse, and rat antibodies | Hamster, mouse, and rat antibodies with recognition of the kappa and lambda chains | Hamster, mouse, rabbit, and rat antibodies | Hamster, mouse, and rat antibodies with recognition of the kappa and lambda chains |
Format | One vial, dispense as a single drop | One vial, dispense as a single drop | One vial positive beads, one vial negative beads | One vial, dispense as a single drop |
Laser compatibility | Compatible with most standard lasers, UV to 633 nm. Improved for polymer dye use from violet laser. | Compatible with most standard lasers, UV to 633 nm | Compatible with most standard lasers, UV to 633 nm. | Compatible with most standard lasers, but not with UV or violet lasers |
Size | 25 tests | 100 tests | |||
Cat. No. | 01-3333-41 | 01-3333-42 | 01-2222-41 | 01-222-42 | A10513 | A10497 | 01-1111-41 | 01-1111-42 |
* More antibody binding compensation beads available. Goat and sheep host species should use single color cell and FMO controls, not beads. |
ArC Amine Reactive Compensation Bead kit (LIVE/DEAD) | |
---|---|
Application | Cell viability assay |
Reactivity | LIVE/DEAD fixable dead cell stains* |
Format | One vial positive beads, one vial negative beads |
Laser compatibility | Compatible with most standard lasers, UV to 633 nm |
Size | 25 tests | 100 tests |
Cat. No. | A10628 | A10346 |
* Also applicable to similar amine reactive dyes |
GFP | mCherry | RFP | CFP | YFP | |
---|---|---|---|---|---|
Application | GFP (Green Fluorescent Protein); labeled beads are present at 3 levels of GFP-like intensity | mCherry (monomeric red fluorescent protein); labeled beads are present at 3 levels of mCherry-like intensity | RFP (Red Fluorescent Protein); labeled beads are present at 3 levels of RFP-like intensity | CFP (Cyan Fluorescent Protein); labeled beads are present at 3 levels of CFP-like intensity | YFP (Yellow Fluorescent Protein); labeled beads are present at 3 levels of YFP-like intensity |
Reactivity | GFP isoforms | mCherry isoforms | RFP isoforms | CFP isoforms | YFP isoforms |
Format | One vial, dispense as a single drop | One vial, dispense as a single drop | One vial, dispense as a single drop | One vial, dispense as a single drop | One vial, dispense as a single drop |
Laser compatibility | 488 nm | 561 nm | 561 nm | 405 nm | 488 nm |
Size | 1 mL (25 tests) | 1 mL (25 tests) | 1 mL (25 tests) | 1 mL (25 tests) | 1 mL (25 tests) |
Cat. No. | A10514 | A54743 | A54740 | A54742 | A54741 |
UltraComp eBeads Plus compensation beads* | UltraComp eBeads compensation beads | AbC Total Antibody Compensation Bead kit* | OneComp eBeads compensation beads | |
---|---|---|---|---|
Application | Immunophenotyping | |||
Reactivity | Human, rabbit, hamster, mouse, and rat antibodies | Hamster, mouse, and rat antibodies with recognition of the kappa and lambda chains | Hamster, mouse, rabbit, and rat antibodies | Hamster, mouse, and rat antibodies with recognition of the kappa and lambda chains |
Format | One vial, dispense as a single drop | One vial, dispense as a single drop | One vial positive beads, one vial negative beads | One vial, dispense as a single drop |
Laser compatibility | Compatible with most standard lasers, UV to 633 nm. Improved for polymer dye use from violet laser. | Compatible with most standard lasers, UV to 633 nm | Compatible with most standard lasers, UV to 633 nm. | Compatible with most standard lasers, but not with UV or violet lasers |
Size | 25 tests | 100 tests | |||
Cat. No. | 01-3333-41 | 01-3333-42 | 01-2222-41 | 01-222-42 | A10513 | A10497 | 01-1111-41 | 01-1111-42 |
* More antibody binding compensation beads available. Goat and sheep host species should use single color cell and FMO controls, not beads. |
ArC Amine Reactive Compensation Bead kit (LIVE/DEAD) | |
---|---|
Application | Cell viability assay |
Reactivity | LIVE/DEAD fixable dead cell stains* |
Format | One vial positive beads, one vial negative beads |
Laser compatibility | Compatible with most standard lasers, UV to 633 nm |
Size | 25 tests | 100 tests |
Cat. No. | A10628 | A10346 |
* Also applicable to similar amine reactive dyes |
GFP | mCherry | RFP | CFP | YFP | |
---|---|---|---|---|---|
Application | GFP (Green Fluorescent Protein); labeled beads are present at 3 levels of GFP-like intensity | mCherry (monomeric red fluorescent protein); labeled beads are present at 3 levels of mCherry-like intensity | RFP (Red Fluorescent Protein); labeled beads are present at 3 levels of RFP-like intensity | CFP (Cyan Fluorescent Protein); labeled beads are present at 3 levels of CFP-like intensity | YFP (Yellow Fluorescent Protein); labeled beads are present at 3 levels of YFP-like intensity |
Reactivity | GFP isoforms | mCherry isoforms | RFP isoforms | CFP isoforms | YFP isoforms |
Format | One vial, dispense as a single drop | One vial, dispense as a single drop | One vial, dispense as a single drop | One vial, dispense as a single drop | One vial, dispense as a single drop |
Laser compatibility | 488 nm | 561 nm | 561 nm | 405 nm | 488 nm |
Size | 1 mL (25 tests) | 1 mL (25 tests) | 1 mL (25 tests) | 1 mL (25 tests) | 1 mL (25 tests) |
Cat. No. | A10514 | A54743 | A54740 | A54742 | A54741 |
eBeads are microspheres that contain a mixture of antibody-coated positive compensation beads and uncoated negative compensation beads, combined in one vial. This set of compensation beads are useful when using many lasers and multiple antibodies from different species.
The advantages of these types of beads include:
Figure 2. Staining of UltraComp eBeads Plus compensation beads with 14 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.
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. These compensation beads produce extremely bright signals.
Each kit offers:
AbC compensation kits are available to recognize either mouse or rat and hamster.
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 Invitrogen Attune Acoustic Focusing Cytometer using 488 nm excitation and a 574/26 nm bandpass filter.
Viability dyes are useful to gate live vs dead cells in flow cytometry experiments. ArC Amine Reactive Compensation Beads were developed to bind Invitrogen LIVE/DEAD Fixable Dead Cell Stains and other similar amine reactive dyes. LIVE/DEAD dyes added to these beads will produce a more similar spectrum as compared to compensating based on the sole fluorophore.
Properties of these beads include:
When using compensation beads for amine reactive dyes, a control with purely dead cells both unstained and stained with LIVE/DEAD can be added to adjust gating.
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.
BrightComp eBeads compensation beads are modified microspheres stained with a dye that has a near-identical spectral match to GFP, mCherry, RFP, CFP, and YFP at 3 levels of intensity. BrightComp eBeads allow for easy compensation of samples with different levels of GFP, mCherry, RFP, CFP, and YFP expression (Figure 5).
These beads offer:
Try these beads with your experiment, and save more of your sample
Figure 5. Multiple intensities of BrightComp eBeads Compensation Beads. Fluorescent proteins can be expressed at varying levels, resulting in the detection of a range of fluorescent intensities. When setting compensation, selecting the bead peak with a higher intensity than the experiment sample is recommended. Data were acquired on a flow cytometer using a 488 nm laser and emission was collected using 525/50 nm filter for GFP, a 561 nm laser and emission using 620/15 nm filter for mCherry, a 561 nm laser and emission using 585/16 nm filter for RFP, a 488 nm laser and emission using 530/30 nm filter for YFP, a 405 nm laser and emission using 440/50 nm filter for CFP.
Below is a general outline of how to use the compensation beads. Use the technical data sheet from the product for detailed protocols.
Step 1: It is necessary for compensation beads to bind the antibody or reagent used in the specific experiment. Use the selection guide to pick the correct compensation bead that binds the species or reagent and can be excited by the appropriate laser.
Step 2: Add the same antibody or reagent used in samples.
Step 3: Vortex or flick to mix. Incubate for 15-30 min in the dark.
Step 4: Wash with the same Flow Cytometry Staining Buffer used in sample staining, then centrifuge, and decant. Resuspend in Flow Cytometry Staining Buffer. Beads are ready to set compensation settings.
Flow Cytometry Panel Builder—Design your flow cytometry panel with this online tool for a simplified, customizable experience to fit your needs.
Download Flow Cytometry Protocols Handbook
Spectral Flow Cytometry Fundamentals
Intracellular Staining for Flow Cytometry How-To Video—for detecting cytokines and intranuclear markers.
Flow Cytometry Learning Center—Access flow cytometry educational resources for better experiment planning and execution.
Flow Cytometry Support Center—Find technical support recommendations for your flow cytometry workflows, including tips for experimental setup and in-depth troubleshooting help.
Flow Cytometry Panel Design Support—Work with one of our technical sales specialists to discuss your experimental needs and guide you through the process.
Not for resale. Super Bright Polymer Dyes are sold under license from Becton, Dickinson and Company.