Compensation Beads (Cat. Nos. 424601, 424602) contain small, synthetic particles capable of binding fluorescently conjugated antibodies. These beads are mixed with inert, non-antibody binding beads. This allows researchers to generate artificial positive and negative fluorescence populations that mimic the heterogeneous populations of cells in a sample that are negative or positive for a given marker. Compensation beads are also used for setting flow cytometer compensation and voltage values. On full spectral flow instruments, they are used to set spectral unmixing parameters.


They can be advantageous to use over biological samples in certain conditions:

  • If there is a known/expected staining outcome, parameter setting with standardized reagents (e.g. compensation beads) can be done. The staining presence/intensity would not be subject to sample variability (e.g. donor variation and variable marker expression).
  • If you have precious or rare samples, compensation beads can be used instead so you don't have to choose whether to use your samples for instrument setup or data analysis. This is particularly valuable for those setting up large flow cytometry panels that require optimization.
  • In some panels, the antigen targets of some antibodies may be expressed at low levels, making it difficult to obtain concrete positive/negative populations. Compensation beads are an excellent alternative in this scenario.

Protocol Overview


  1. Prior to use, vortex beads vigorously to disrupt aggregates that may have formed during storage.
  2. Add one drop of beads from each bottle (positive and negative) to prepared tubes.
  3. Add antibodies at desired concentration.
  4. Mix and incubate at room temperature for 15-20 minutes, protected from exposure to light.
  5. Wash beads with Cell Staining Buffer (Cat. No. 420201) or equivalent.
  6. Centrifuge at 350 x g for 5 minutes and re-suspend in Cell Staining Buffer or equivalent.

BioLegend Compensation Beads Binding of Common Human, Mouse, Rat, and Hamster Immunoglobulin Isotypes



BioLegend Compensation Beads stained with 23 different FITC-conjugated isotypes of antibodies of mouse, rat, rabbit, donkey, hamster and human origin. Beads were stained with 0.05 µg of antibody per test.

Scatter Profile Comparison



Overlaid scatter profile for BioLegend Compensation Beads (purple) and UltraComp eBeads™ Plus (orange).


Bead Staining With Brilliant Violet 421™ and Specialized Fluorophore-Specific Buffers



BioLegend Compensation Beads were stained with Brilliant Violet 421™ anti-human CD41 Antibody (0.05 µg, Mouse IgG1, κ isotype) in Cell Staining Buffer (purple) or in BD Horizon™ Brilliant Stain Buffer (green).


Temperature Stability Comparison



BioLegend Compensation Beads kept either at 4 °C or 37 °C for one or two weeks, then stained with the indicated antibodies.



14-Color Panel Staining Profile Comparison



14 single-color controls were prepared using either BioLegend Compensation Beads or UltraComp eBeads™ Plus using respective recommended protocols. Unmixing matrices were generated on a 5-laser Cytek™ Aurora using single-color control sets with respective compensation beads (unstained cells were used as background control for each). Overlaid plots shown are human peripheral blood mononuclear cells stained with the full 14-color panel, analyzed with unmixing matrix generated using BioLegend Compensation Beads (purple) or UltraComp eBeads™ Plus (orange). See table below for information on antibodies used and single-color control setup.


Single-Color Controls Table for the 14-Color Panel

Marker Clone Isotype Fluorophore Antibody used (µg)
CD3 UCHT1 Mouse IgG1, κ Alexa Fluor® 700 2.5
CD4 OKT4 Mouse IgG2b, κ PE/Cyanine7 0.375
CD8 RPA-T8 Mouse IgG1, κ PerCP/Cyanine5.5 0.5
CD11b ICRF44 Mouse IgG1, κ Brilliant Violet 711™ 0.5
CD14 HCD14 Mouse IgG1, κ APC/Cyanine7 2.0
CD16 3G8 Mouse IgG1, κ APC 0.75
CD19 HIB19 Mouse IgG1, κ PE/Dazzle™ 594 0.25
CD25 M-A251 Mouse IgG1, κ Brilliant Violet 421™ 0.25
CD27 M-T271 Mouse IgG1, κ Brilliant Violet 510™ 0.4
CD45RA HI100 Mouse IgG2b, κ FITC 1.0
CD45RO UCHL1 Mouse IgG2a, κ Brilliant Violet 605™ 0.5
CD56 HCD56 Mouse IgG1, κ PE 0.4
CD62L DREG-56 Mouse IgG1, κ Brilliant Violet 650™ 0.4
HLA-DR L243 Mouse IgG2a, κ Brilliant Violet 785™ 0.4

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