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Controls in flow cytometry

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Controls are vital in any experiment to reliably distinguish your results from background variation and non-specific effects.

Here we will discuss some essential controls for flow cytometry you must consider to ensure publication quality flow cytometry data.

Unstained controls

The first thing to identify in flow cytometry is your cell population by its forward and side scatter characteristics. After this you need to determine where the negative population will be. To do this you should always have an unstained population. This will allow you to determine the level of background fluorescence or autofluorescence and set your voltages and negative gates appropriately.

Isotype controls

The use of isotype controls in flow cytometry is controversial and divides researchers. In flow cytometry, background levels of staining can be a problem especially with rare populations, cells with low expression levels and when building multicolor panels. Isotype controls have been developed for surface staining and their role is to ensure the observed staining is due to specific antibody binding to the target rather than an artefact. They should not to be used to determine positive versus negative cells or to set gates, and may not be suitable for intracellular staining. To find out more about how isotype controls can be used in your flow cytometry experiments, when to use them and which ones to use, go to our dedicated isotype controls webpage.

Single staining and compensation controls

When performing multicolor flow cytometry, single stained samples are essential to determine the levels of compensation. Single staining will reveal the level of spectral overlap between different fluorophores and allow you to remove or compensate for this overlap. This can be seen in Figure 2 a where the fluorescence of FITC can be detected in the PE channel. Figure 2 b shows how the data looks when properly compensated. This spectral overlap should be compensated for every fluorophore used. Important things to consider, when using single stained samples for compensation, are that the fluorophore needs to be the same and its brightness similar to the one used in your experiment and you need to collect enough events to be statistically significant. For more detailed information on fluorophores and compensation check our Flow Cytometry Basics Guide available to download or as a hard copy.

Viability controls

The presence of dead cells in your sample can greatly affect your staining and therefore the quality of your data. This is because dead cells have greater autofluorescence and increased non-specific antibody binding, leading to false positives and reducing the dynamic range. This may make identification of weakly positive samples and rare populations difficult. Whilst using gates based on the forward and side scatter can help to remove debris and dead cells it will not remove them all. Types of viability dyes to identify dead cells include DNA dyes such as ReadiDrop™ Propidium Iodide or ReadiDrop™ 7-AAD, or protein binding dyes such as VivaFix™ which are fixable for added convenience.