Flow Cytometry Compensation 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 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:

• 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

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 Spectral Unmixing Beads are designed for spectral and conventional flow cytometry, offering flow cytometry controls. These specialized beads bind to fluorophore-conjugated antibodies, delivering precise single-color spectral unmixing controls. They are compatible with mouse, rat, hamster, rabbit, and human (including recombinant) antibodies, offering broad versatility. Advantages of these spectral unmixing and compensation beads include:

• Unmixing and spectral performance comparable to real cells
• Reduced fluorescence background noise for enhanced signal detection
• Bright and consistent fluorophore signals
• Compatibility with more species
• Improved unmixing accuracy yielding more reliable results (see Figure 2)

UltraComp Spectral eBeads are suitable for use with fluorophores excited by ultraviolet (355 nm), violet (405 nm), blue (488 nm), green (532 nm), yellow-green (561 nm), or red (633–640 nm) lasers.

Each drop contains two populations of beads: a positive population that binds with any mouse, rat, hamster, rabbit, or human antibody, and a negative population that does not bind to any of the antibody conjugates. This bimodal distribution is ideal for setting up single-color controls for unmixing and compensation in multicolor and high-parameter flow cytometry experiments (Figure 2).

Figure 2. Comparing UltraComp Spectral eBeads and UltraComp Plus eBeads on unmixing performance. PBMCs stained with anti-CD4 antibodies in a 33-color, single stained control panel were unmixed either with UltraComp eBeads Plus Compensation Beads (top) or UltraComp eBeads Spectral Unmixing Beads (bottom). Example dot plots for small molecule dyes, red emitting dyes, and tandem dyes from each compensation bead panel are shown to highlight the improved unmixing performance of the UltraComp Spectral Unmixing eBeads across different fluorophore combinations as seen by the closer alignment of the medians of the negative (black dotted line) and positive (red dotted line) populations. Data were collected and analyzed using the Cytek Aurora flow cytometer.

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:

• 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 3. 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.

Learn more: UltraComp compensation beads protocols for flow cytometry

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:

• Distinct positive and negative populations of beads that can be used to set compensation: 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, which is helpful when using antibodies conjugated to very bright fluorophores like PE
• Reagents that 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.

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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
• The ability to 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.

Add ArC Amine Reactive Compensation Beads today

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 6).

These beads offer:

• Consistent, accurate, and simple-to-use reagents for setting flow cytometry compensation when using GFP, mCherry, RFP, CFP, or YFP
• 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 6. 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.

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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.

1. Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition). Eur. J. Immunol., 49: 1457-1973.
2. Compensation in Flow Cytometry. Current Protocols in Cytometry, 22: 1.14.1-1.14.20.

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