Flow Cytometry diagram
Credit: Doug Auld, PhD/Novartis Institutes for BioMedical Research

Flow cytometry provides a useful method by which scientists can measure and analyse different properties of individual cells, including size, internal complexity (granularity) and the presence of cell surface and intracellular markers using fluorescence. Flow can help identify individual cell subpopulations within a homogenous or heterogenous mix, confirm cell purity, and measure changes to cells following treatment under experimental conditions. Applications range from basic and preclinical research to human disease diagnoses and therapies, including the production of protein- and cell-based therapies (1).

As an invaluable tool with a wide breadth of applications, having reproducible and reliable results is critical, including routine calibration and the careful use of standard controls (2). This article will review essential basic concepts for testing flow cytometer performance that can help promote success.

Starting out: Instruments and standards

Starting out with a reliable instrument is an important first step, as even the most sensitive instrument will not be of much help if it constantly requires maintenance. Background noise arises when random photons of light or electron emissions generate photocurrents that are picked up by the photomultiplier tube at the interface of the fluidics and electronics system in flow cytometers. If selecting a new flow cytometer, instruments with low noise generally provide better resolution than instruments with higher background noise. Setting an optimal threshold reduces background noise in the flow cytometer.

Even after the right instrument has been installed, however, regular maintenance should be carried out as per the guidance and documentation provided with each instrument (3). Once a flow cytometer is ready for use, standard controls can be employed to confirm that it is properly calibrated and running as expected, while bearing in mind the five critical parameters for accurate flow results.

“Precision, sensitivity, reliability, reproducibility, and accuracy are the most important factors to consider when testing the performance of a flow cytometer,” says Michelle Scott, Senior Scientist at Bio-Rad Laboratories. “For testing day-to-day instrument performance, fluorescent beads are the most commonly used standard,” Scott advises.

Precision is measured as the coefficient of variation (CV), which describes the extent to which identical or nearly identical particles such as fluorescent beads are measured as identical values by a flow cytometer. Sensitivity is the minimum size of particles or amount of fluorescence that a flow cytometer can distinguish above background noise and is often measured as molecules of equivalent soluble fluorochrome (MESF). The degree to which results produced by a flow cytometer reflect the actual values of the variables measured is the accuracy of the instrument which can be affected by factors such as detector non-linearity. And finally, reproducibility is the ability of a flow cytometer to maintain the same precise, sensitive and accurate measurements over time.

Many commercially available beads are polymer-based hard-dyed microspheres that can be used to monitor performance at multiple wavelengths (1,3). Beads provide useful tools for comparing signal intensities, often including multiple peaks over time. Each type of bead-standard corresponds to a certain number of fluorophore molecules in solution, based upon its fluorescent intensity. For clinical applications such as CD4+ cell counting, specific set-up requirements will be in place as well as the potential for specific beads and software (1).

Another generally accepted universal standard is the measurement of chicken erythrocyte nuclei fluorescence using a DNA binding dye, which can be helpful for measuring cellular DNA content. Other standard cell types include rainbow trout erythrocytes and human lymphocytes (1).

In terms of the frequency of flow cytometer testing with standards that is generally required for optimal performance, measuring precision and instrument sensitivity should be carried out daily, in reference to a known bead value. Scott emphasizes that detecting changes in the trends can help identify potential issues early, such as laser misalignment. By contrast, testing sensitivity via absolute molecules of equivalent soluble fluorochrome (MESF) or the linearity of the instrument is not necessarily required on a day-to-day basis. Regular monitoring of optical alignment, fluidics, laser power and PMT voltages is also highly recommended (3).

Troubleshooting tips

“For a general user rather than a flow core specialist, the most common issues relate to performance of the instrument versus sample preparation,” says Scott. Unexpected results may arise from poor or improper instrument performance or set-up, or alternatively due to poor sample preparation.

The forward scatter (FSC) and side scatter (SSC) voltages used to visualize samples can also prove to be problematic for general flow users and therefore need to be optimized for every sample. Setting up photomultiplier tube (PMT) voltages for fluorescence detection should be carried out, using unstained cells or by voltration, an experiment that uses a mixture of negative and labelled beads to optimize PMT voltage. Optimal voltage range gives the maximum separation between particle populations while retaining signals in linear detection range.

The stability of the detection system can also be monitored via PMT voltage or detector gain measurements, using the same PMT voltage or gain settings to confirm that FSC and SSC signals are stable when calibrated against bead-based standards (1). Proper placement of optical filters is also important, as is having a standard method for identifying the potential causes of unexpected results, including sample preparation.

Scott concludes with a key piece of advice: “Proper controls are key.” Use of negative, compensation and “fluorescence minus one” (FMO) controls should always be used alongside samples, in addition to beads for routine instrument testing. Regular testing, coupled with the application of best practice guidelines and use of the right controls, can improve the consistency and reproducibility of flow cytometry data.



  1. Wang L., Hoffman R.A. (2017) Flow Cytometer Performance Characterization, Standardization, and Control. In: Robinson J., Cossarizza A. (eds) Single Cell Analysis. Series in BioEngineering. Springer, Singapore.
  2. Hoffman, R.A., Wood, J.C.S., 2007. Characterization of Flow Cytometer Instrument Sensitivity. In: Current Protocols in Cytometry, John Wiley & Sons, Inc., USA.
  3. van der Strate, B., et al., 2017. Best practices in performing flow cytometry in a regulated environment: feedback from experience within the European Bioanalysis Forum. Bioanalysis 9(16):1253-64.
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