Recommended controls for flow cytometry

When setting up your flow cytometry test, make sure you include the appropriate controls for:

• Cell viability: Dead cells can produce artifacts due to non-specific binding and autofluorescence,potentially leading to inaccurate results

• Autofluorescence: Naturally occurring cell components, such as NADPH and flavins, can emit fluorescence that may mask antigen-specific signals

• Spectral overlap: Fluorescence emitted from one fluorophore may also be detected on a different channel, significantly affecting measurements on a given channel

• Undesirable antibody binding: This occurs when the antibody binds to either an off-target epitope, Fc receptor (FcR), or cellular components through its conjugated fluorophore. FcR binding can be reduced with the addition of specific blocking reagents before staining

It is essential to eliminate dead cells from your flow cytometry data analysis because they can give rise to false positives due to autofluorescence and increased non-specific binding.

Several markers are available that can distinguish between dead and live cells. As some of these dyes bind DNA, they may also be used for DNA content or cell cycle analysis.

Cell impermeable dyes such as 7-Aminoactinomycin D (7-AAD), propidium iodide, Nuclear Green DCS1, or DRAQ7™ are used on unfixed cells. These dyes discriminate between live and dead cells by staining only dead cells and being actively excluded from living ones.

An alternative method for determining cell viability is to use the cell-permeable fluorescent dye calcein AM. This dye is hydrolyzed to green fluorescent calcein by intracellular esterases in living cells. Cells stained with this dye can also be fixed with paraformaldehyde and then analyzed. 

Cell type and physiological conditions influence autofluorescence. Naturally occurring cell components, such as NADPH and flavins, can emit fluorescence upon 488 nm wavelength laser excitation. 

To check if autofluorescence presents a problem in your experiment, analyze an aliquot of unstained cells on the flow cytometer using the same cell treatment and machine settings as the experimental sample. If there is significant autofluorescence, using a different laser wavelength can resolve the problem. Refer to our fluorophore chart or multicolor selector to explore alternative lasers using your dye of choice.

When carrying out a multicolor flow cytometry experiment, the emission spectra of the various fluorophores can overlap, resulting in detection in a different channel (also called spillover). 

This phenomenon can result in false positives or incorrect gating when positive or negative boundaries are ambiguous. However, this can be controlled with compensation, where spectral overlap is estimated and subtracted from the total detected signal to yield an estimate of the actual amount of each dye, or including fluorescence minus one (FMO) controls to define the positive/negative populations.

FMO controls, samples stained with all antibodies in a panel except for one, are essential for providing a measure of spillover in a given channel and accurately discriminating positive and negative cell populations.1 This control provides a true negative control as it considers how the other fluorophores in your panel affect the signal observed in the channel used for the examined fluorophore. For example, in a multicolor panel of FITC, PE-Cy5, PE-Cy7, and PE, the PE FMO control would contain the FITC, Cy-PE, and Cy7-PE reagents, but not the PE (Figure 19).

This broad term includes every instance of antibody binding that prevents correct interpretation of the data. Optimizing your staining protocol and running appropriate negative controls can help to detect and alleviate these effects.

Antibodies are key components of flow cytometry techniques, yet many have not been validated for specificity, lack of cross-reactivity, or use in flow cytometry applications.² To ensure reproducible, robust data, it is important to either validate your antibodies or purchase validated antibodies from a trusted supplier. 

Non-optimal antibody concentrations can increase non-specific binding or reduce the sensitivity of the measurement. Therefore, titrate all antibodies to determine the best signal-to-noise ratio (Figure 20).

Phagocytic cells, such as monocytes, have Fc receptors (FcRs) on their surface that can bind nonspecifically to the Fc region of antibodies—adding FcR blocking reagents before staining can block this binding (Figure 21). You should also include this blocking step in homogenized tissue samples, which may contain macrophages, as well as cell culture lines such as Daudi and THP-1.

The negative control should be a population of cells that do not express the antigen of interest, ideally a knock-out cell line. This sample should be exposed to the same experimental conditions as the population in the study. Use this control to set gating regions and discern positive from negative cells.

An isotype control is an antibody raised against an antigen not present on or in the analyzed cell type. Isotype controls determine the level of background fluorescence caused by non-specific antibody binding. They should not be used to distinguish positive from negative cells or set positive gating regions.

An ideal isotype control should:

• Match the primary antibody in host species, class and subclass of heavy and light chains, fluorophore type, and number of fluorophore molecules per immunoglobulin
• Be derived by the same manufacturing process and presented in the same formulation

Refer to our guide to selecting isotype controls for more information.

The isoclonic control shows whether a fluorophore or other antibody conjugate is binding non-specifically to cellular components. 

Cells are stained with the conjugated antibody in the presence of an excess of identical (isoclonic) unlabeled antibody. Specific antibody binding sites in the sample are taken up by the unconjugated antibody, while the conjugated antibody can only bind through the conjugate. 

A lack of fluorescent signal suggests that the conjugate is not binding non-specifically to any components within the sample. As with any isotype control, this type of control is solely qualitative.