What are compensation beads?
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. Beads are recommended when:
- Multiple fluorophore emissions overlap in the same detector (Figure 1)
- Poorly expressed markers do not express a large distinction between positive and negative populations
- Limited amount of sample is available to setup/run controls and collect enough events for meaningful data
- Creating large multicolor immunophenotyping panels to set accurate single-color compensation
- When performing multiple plates or large experiments, bead controls will help with standardization and save sample
Use the table below to determine which compensation bead is correct for your experiment.
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Compensation bead selection guide
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* | AbC Total Antibody Compensation Bead kit* | |
|---|---|---|
| Application | Immunophenotyping | Immunophenotyping |
| Reactivity | Human, rabbit, hamster, mouse, and rat antibodies | Hamster, mouse, rabbit, and rat antibodies |
| Format | One vial, dispense as a single drop | One vial positive beads, one vial negative beads |
| 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. |
| Size | 25 tests | 100 tests | 25 tests | 100 tests |
| Cat. No. | 01-3333-41 | 01-3333-42 | A10513 | A10497 |
| * 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 BrightComp eBeads compensation beads | |
|---|---|
| Application | GFP (green fluorescent protein). Labeled beads are present at 3 levels of GFP-like intensity. |
| Reactivity | GFP isoforms |
| Format | One vial, dispense as a single drop |
| Laser compatibility | 488 nm |
| Size | 25 tests |
| Cat. No. | A10514 |
| UltraComp eBeads Plus compensation beads* | AbC Total Antibody Compensation Bead kit* | |
|---|---|---|
| Application | Immunophenotyping | Immunophenotyping |
| Reactivity | Human, rabbit, hamster, mouse, and rat antibodies | Hamster, mouse, rabbit, and rat antibodies |
| Format | One vial, dispense as a single drop | One vial positive beads, one vial negative beads |
| 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. |
| Size | 25 tests | 100 tests | 25 tests | 100 tests |
| Cat. No. | 01-3333-41 | 01-3333-42 | A10513 | A10497 |
| * 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 | |
New! UltraComp eBeads Plus compensation beads
Are you using more polymer dyes from the violet and UV lasers? Are you building a bigger panel and need accurate compensation?
Fluorescence compensation controls and beads
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 to 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:
- Unstained sample can assess autofluorescence.
- FMOs evaluate the effect of other fluorophores and compensation on background.
- All experiments must use single-color controls such as compensation beads to set gating parameters and optimize voltages for positive and negative signals.
Unstained samples and single-color controls are needed for setting parameters on spectral analyzers. Compensation beads are useful when they are as bright or brighter than samples used in a panel and when the fluorochrome spectrum are identical between sample and beads.
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.
How to use compensation beads?
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.
- If using a primary antibody bound to a secondary antibody conjugated to a fluorophore, use only the secondary antibody.
- Do not use similar fluorophores because they are spectrally different and will not properly compensate.
- Prepare beads fresh for each time sample is run.
- Beads require less antibody or reagent than cells. Titrate your antibody or reagents before starting your experiment. It is recommended to start with 1/100 of the amount of antibody or reagent used in the sample.
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.
Beads for compensating flow cytometry antibodies
UltraComp and OneComp eBeads for compensation
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 beads include:
- Easily expand your panels and retain the cells you need since eBeads have low autofluorescence and provide signal sensitivity
- Designed for ease of use with combined positive and negative beads in one vial and dispense as a single drop
- Binds a wide range of species and is excited by most lasers
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 Total Antibody Compensation kit
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 signal. Each kit offers:
- 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.
- Very bright positive signal. This is helpful when using antibodies conjugated to very bright fluorophores like PE.
- Can be combined with other compensation beads including ArC Amine Reactive Compensation Beads.
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.
Beads for compensating LIVE/DEAD dyes
ArC Amine Reactive compensation beads
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:
- 2 drop kit for accurate and bright positive signal.
- Combine the AbC Total Antibody Compensation Bead kit and ArC Amine Reactive Compensation Bead kit together to determine compensation in multicolor immunophenotyping experiments.
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.
Beads for GFP compensation
GFP BrightComp eBeads Compensation Beads
The GFP BrightComp eBeads compensation beads are modified microspheres stained with a dye that has a near-identical, spectral match to GFP at 3 levels of intensity (Figure 5). The product includes modified microspheres to allow for easy compensation of samples with different levels of GFP expression (Figure 6). These beads provide:
- Consistent, accurate, and simple-to-use reagents for setting flow cytometry compensation when using green fluorescent protein (GFP).
- Ease-of-use with a single drop containing both negative and positive beads.
Try these beads with your experiment, and save more of your sample
Figure 5. GFP BrightComp eBeads Compensation Beads can be used to compensate for GFP. (A-C) HeLa cells, a human cervical cancer cell line, were transduced with CellLight Histone 2B-GFP, BacMam 2.0 resulting in GFP expression. Cells were subsequently harvested and stained with rat anti-human/mouse CD44-PE and analyzed on a flow cytometer. Data was acquired on a flow cytometer using a 488 nm laser and emission was collected using 525/50 nm and 585/42 nm filters. Samples were auto-compensated. Samples where GFP BrightComp eBeads compensation beads were used to compensate (C), show the same degree of compensation as when compared to samples compensated using the GFP-expressing HeLa cells (B).
Figure 6. Multiple intensities of GFP 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, selection of 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 for GFP.
Resources
Flow Cytometry Learning Center—Access flow cytometry educational resources for better experiment planning and execution.
Flow Cytometry Panel Builder—Design your flow cytometry panel with this online tool for a simplified, customizable experience to fit your needs.
5 Steps Resources
Support
Flow Cytometry Support Center—Find technical support recommendations for your flow cytometry workflows, including tips for experimental setup and in-depth troubleshooting help.
Not for resale. Super Bright Polymer Dyes are sold under license from Becton, Dickinson and Company.
For Research Use Only. Not for use in diagnostic procedures.
