Performing Compensation for Flow Cytometry Simplified with These Compensation Beads

July 01, 2019

BlueskyReddit
Memorial University of Newfoundland
Division of BioMedical Sciences, Faculty of Medicine
Research Assistant

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Company:

BD Biosciences

Product Name:

Anti-Mouse Ig, κ/Negative Control (FBS) Compensation Particles Set

Catalog Number:

552843

Compensation is a necessary component of multi-color flow cytometry experiments. The spillover of emitted fluorescence of individual fluorophores into channels that are not being used to detect that particular fluorophore is necessary in order properly compensate and thus analyze your flow cytometry data. Single stained control samples are used calculate this compensation. Antibody capture beads (or compensation particles/beads) offer many advantages over cells for performing these single stained controls. There are several compensation bead kits on the market from a variety of manufacturers; however this product was chosen because it works with a wide range of antibodies used for staining in our flow cytometry experiments.

Experimental Design and Results Summary

Application

Flow Cytometry

Starting Material

The product, staining buffer for flow cytometry, centrifuge, fluorophore-conjugated antibodies used for multi-color flow cytometry, flow cytometer

Tips

Check to make sure that these beads will bind your antibody of interest (i.e., your staining antibody must be mouse Ig, kappa light chain). You can use less antibody and beads per single stain control than the recommended protocol suggests; however, you will have to play around with this in order to achieve sufficient brightness. You may want to consider staining these beads in the same manner that you stain your cells, especially if fixation/permeabilization is performed after cell staining, in order to avoid any differences between your samples used for compensation and your test samples.

Results Summary

The recommended protocol in the technical data sheet was followed for bead staining, except that approximately 1/2 a drop of each bead was used for each single stain control and the amount of antibody used for bead staining was ~1/5 of that used for staining of fluorescence minus one controls and cell samples. Several single stained controls were performed with antibodies conjugated to FITC, PE, BV421, BV510, BV650, or AlexaFluor647. Only one example is shown. As shown in the figure, two distinct populations of beads are observed (FITC- and FITC+). In this example, the FITC+ population was sufficiently bright (brighter than cell samples) to perform compensation even though only ~1/5 the amount of antibody used for staining cells was used to stain the beads. Similar results were obtained for all other fluorophores tested. This data was obtained using a Beckman Coulter MoFlo Astrios EQ cell sorter.

DOI or PMID #

N/A

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Summary

The Good

Ease of use. Standard protocol. Can use less antibody to obtain single stained controls for compensation than when using cells. Saves your previous tissue samples.

The Bad

Does not work will all fluorophore-conjugated antibodies used for staining for flow cytometry...check the species and immunoglobulin light chain composition of your flow cytometry antibodies.

The Bottom Line

This is an excellent product for performing singe stained controls for compensation calculations for flow cytometry experiments. If you are working with valuable tissue samples where the amount of tissue/cells is a limiting factor, using this reagent for compensation will help save on your sample. Just be sure that the antibodies that you are using for staining are compatible with these beads.

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