Automated compensation for multicolor flow cytometry

Multicolor flow cytometry is commonly used to acquire large quantities of information on several cell subpopulations from a single sample, and instrument compensation is a crucial part of this process. MACSQuant® Analyzers offer fully automated compensation with single-stained cells or compensation beads. On this page, you’ll find a compilation of resources to make your compensation simple, convenient, and successful.  


Automated flow cytometric compensation with the MACSQuant Analyzer Family

Free your lab staff from unnecessary tasks so they can get on with their research: the MACSQuant Analyzer Family boasts automated compensation (and more) to save time, money, and to reduce opportunities for human error. It’s as simple as press play and walk away.

Why perform compensation?

Multicolor flow cytometry is one of the most commonly used techniques for acquiring a large quantity of information on several cell subpopulations from a single sample. Good panel design helps get more information from your sample.

We can help you understand the basics of multicolor flow cytometry and suggest simple guidelines in creating sophisticated panel designs. Try our panel builder tool to see panel optimization in action, and for help in choosing the right antibodies.

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Online tutorial

Understanding the basics of multicolor flow cytometry

Join Dr Guerric Epron in his webinar on the basics of flow cytometry, including fluorescence, compensation, data-spread effect, and panel design.  

Automated compensation on MACSQuant Instruments

The MACSQuant Analyzers support your multicolor flow cytometry with several automated features, from housekeeping to standardized analysis. They can assist with an easy set-up for manual compensation, or with truly hands-free and fully automated compensation. Our online tutorial gives you insights on the important details of setting up an optimal compensation workflow on digital analyzers, including the helpful automated compensation feature.

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Online tutorial

Compensation on spectral overlap in flow cytometry

Learn how to set up compensation workflows on the MACSQuant Analyzer, including the use of MACSQuantify™ Software. Join Dr. Frank Hardung in his webinar on the compensation of spectral overlap in flow cytometry

Flow cytometer compensation is often considered a tedious and time-consuming procedure that requires a significant degree of experience and expertise to do right. MACSQuant Instruments completely automate this procedure, making for reliable and reproducible results.

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Application note

Performing automatic compensation on the MACSQuant Flow Cytometer Family

Learn how to use either single-stained cells or compensation beads with MACSQuant Instruments in an accurate and reliable, automated procedure. In this application note, 8-color automated compensation using peripheral blood mononuclear cells is detailed for the MACSQuant Analyzer 10.

Compensation using MACS Comp Beads

For automated or manual compensation after staining with fluorochrome-conjugated antibodies, MACS Comp Bead Kits can be used achieve optimal acquisition and display of data obtained by flow cytometry. Instead of using cells to set up the compensation, compensation beads can be used. These act as “artificial cells” that can be stained with fluorochrome-antibody conjugates.

After preparing single stainings by loading capture beads with the fluorochrome-antibody conjugates used in the experiment, these positive beads are capable of providing a very bright fluorescence emission. Along with the negative (blank) beads included, they function as an excellent compensation control. Negative and positive beads have the same autofluorescence and thus the capture beads can be loaded with the same fluorochrome conjugate lot used in the experiment. They also enable lot-specific compensation of tandem fluorochromes. An example of compensation with a MACS Comp Bead Kit is shown in the figure below:

Before compensation
After compensation

The MACS Comp Beads – anti-mouse Igκ were labeled with antibodies conjugated to FITC and PE and analyzed. Gating was performed on single bead events (A). MACS Comp Beads are shown before (B) and after (C) compensation using the Compensation Multicolor program with the MACSQuant Analyzer.

A number of kappa light chain-based kits are available from Miltenyi Biotec, enabling straightforward compensation of fluorochrome-antibodies of murine, human, or rat origin. Our REAfinity™ Recombinant Antibody Portfolio is also well worth looking into in this regard.

Laboratory lifehack

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Laboratory lifehacks: automated compensation for flow cytometry

There’s no doubt about it: flow cytometry can be repetitive, and at times frustrating. We’ve got you covered.

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