Flow Cytometry Controls - Quick Tips for Better Flow Cytometry Data

Flow cytometry controls for better data

The goal in a flow cytometry experiment is often to measure which cells are positive or negative for a given marker, and the precise ratio of positive to negative cells. Like in all scientific measurements, proper controls are a must to ensure that the signals and backgrounds are correctly measured and analysed.  Depending on the experiment, multiple controls may be needed to understand and eliminate the different sources of background.

Controls in flow cytometry experiments can either be related to instrument settings or may be experiment-associated.

Instrument setting controls

Certain aspects of cytometer setup like laser alignment, laser time delay, sensitivity, etc need only to be checked periodically, and not before every experiment. Depending on the markers and fluorochromes used, certain setup controls can vary between the type of flow cytometer (analog vs digital) or the experiment (single-colour or multicolour).¹

Multicolour flow cytometry experiments require the setting of the instrument gain (photomultiplier tube voltages), determining the degree of spillover and the required compensation for the experiment. ¹

The BD™ Cytometer Set-up and Tracking (CS&T) beads are designed for use on BD™ flow cytometers running BD FACSDiva™ Software to provide a standardised method to perform quality control of the instrument’s optics, electronics and fluidics.

Experiment-associated flow cytometry controls

Experiment-associated controls involve checking for the different sources of background to set an appropriate gate to analyse the cell population of interest. Some of the experiment-associated controls discussed here are single-stain controls, FMO (fluorescence minus one) controls, isotype controls and biological controls.

Single stain controls

These are controls that are used to measure the spillover of one fluorochrome’s emission spectra into another detector. The data collected in such cases must be ‘compensated’ to remove the effects of the spillover. A common method of estimating compensation involves the use of a series of beads or cell samples, each stained with a single fluorescent marker used in the panel. The correct compensation can be calculated and applied by the software either in real time during acquisition or afterward during analysis.

FMO controls

While compensation controls for unwanted signals into a secondary detector, it does not correct background spread due to spillover. FMO, or fluorescence minus one control, involves the use of all the antibodies in the panel minus the one for which the appropriate gate has to be determined. The data obtained using an FMO control can thus, help determine the background from all other fluorochromes in the detector that is being used to measure the target fluorochrome.

BD provides a huge range of single-colour antibodies that can be used as single stain and FMO controls.

Isotype controls

The antibody of a marker under study can also bind in a non-specific way to our cells of interest, resulting in noisy data. FMO controls cannot be used to measure this as, by definition, they lack the antibody of the marker being studied. So, isotype controls are used to mimic the non-specific binding of an antibody conjugated to a particular fluorochrome. However, matching the isotype of the test antibody is important. Isotype controls have two major limitations. The first is that as the background depends on several factors, it is difficult to find an isotype control with the same background as the test antibody.¹ The second limitation is by themselves, isotype controls do not account for fluorescence spillover from other channels.

Learn more about how to set isotype controls for flow cytometry.

Biological controls

Biological controls are often very appropriate to set boundaries between positive and negative expression events. For example, in stimulation assays, the unstimulated sample usually provides the best means to distinguish between the negative and positive expressions. There may be exceptions where a high background in the unstimulated sample makes it more difficult to set a clear positive/negative boundary but in most cases, this control is far more relevant than an isotype or FMO control.

Conclusion

The choice of the most appropriate controls in flow cytometry is experiment-dependent. It is advisable to run several types of controls during assay development to determine the main source of background and the best strategy to account for it in a given experiment.

Check out our BD Biosciences flow cytometry protocols for your experiments.