Molecules of Equivalent Soluble Fluorochrome (MESF) Beads for Flow Cytometry

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Overview

Flow cytometry is a powerful tool, but without standardization, fluorescence data can be difficult to compare across experiments, instruments, or time. There are several aspects to effective flow cytometry standardization, one of which is including MESF beads in experimental workflows. When validating assays, monitoring instrument performance, or publishing peer-reviewed data, MESF beads allow the move from relative fluorescence, such as median fluorescence intensity (MFI), to absolute quantification.

What Are MESF Beads?

MESF beads are calibration particles embedded with a known number of fluorophore molecules. Each bead population corresponds to a specific MESF value, allowing the:

• Creation of standard curves for fluorescence quantification
• Normalization of data across instruments and time points
• Report of fluorescence values in standardized units for reproducibility
• Instrument fluorescence detection threshold and instrument linearity

Why Use MESF Beads?

MESF beads are applicable in a variety of situations, as described in Table 1. Quantitative data reporting for fluorescent values is becoming more desirable for peer-reviewed publications; it makes the findings more robust and easier for the scientific community to interpret and replicate. For example, the guidelines for publishing flow cytometry results on extracellular vesicles (EVs) as outlined in The Minimum Information for Studies of Extracellular Vesicles (MISEV) and MIFlowCyt-EV recommend reporting in MESF values.

Table 1. Benefits of using MESF beads.

How to Use MESF Beads

Using MESF beads is a simple three-step process:

1). Run the beads and your sample, which is labeled with the same fluorophore as the beads, on your flow cytometer using the same settings for both the beads and sample.

2). Plot a standard curve of median fluorescent intensity (MFI) versus MESF value for the bead populations.

3). Use this curve to determine the MESF value from your sample's MFI.

If MESF sample values differ between cytometers, check each instrument; confirm correct experimental setup, such as the correct detection filter is used, and use QC beads to confirm that each cytometer is operating within its expected range.

Differences may also result from lot-to-lot variation of reagents. If no issues are identified, data can be normalized to match MESF values across instruments.

Best Practices for Reliable Results

To get accurate data the most important factor is to maintain consistent instrument settings, such as laser power, and photomultiplier tube (PMT) or avalanche photodiode (APD) voltages should not be altered between running the calibration beads and samples of interest. Also, to obtain accurate readings, fluorescent samples should fall within the standard curve range.

MESF Range

Bio-Rad has three different MESF bead kits available (Table 2) for PE and FITC fluorophores. MESF beads help you achieve precision, consistency, and confidence in every experiment.

Table 2. MESF range and description.