All tags Flow cyt Recommended controls for flow cytometry

Recommended controls for flow cytometry

​​Improve your flow cytometry results by using the appropriate controls.

When ​​​setting up your experiment, make sure you include the following controls:

  • Cell viability. Dead cells can produce artefacts due to non-specific binding and increasing autofluorescence levels, potentially leading to erroneous conclusions.
  • Autofluorescence. Naturally occurring cell components such as NADPH and flavins can emit fluorescence that may mask antigen specific signal.
  • Spectral overlap. Fluorescence emitted from one fluorophore can be detected in a different channel. This phenomenon can seriously affect measurements on a given channel. 
  • Undesirable antibody binding. Undesirable antibody binding. This occurs when the antibody binds to either an off-target epitope or an Fc-receptor (but not as a receptor-ligand interaction), or binds through to an epitope or antigen through its conjugated fluorophore.

Cell viability

Dead cells can give rise to false positives due to autofluorescence and increased non-specific binding. It is important to eliminate these dead cells from your data analysis.

​​Several markers are available that can be used to 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 dead cells and being actively excluded by live cells. 

Calcein AM is a cell-permeable fluorescent dye for determining cell viability. This dye is hydrolyzed to green-fluorescent calcein by intracellular esterases in live cells. Cells stained with this dye can also be fixed with paraformaldehyde and then analyzed.

We provide a wide range of cell viability markers:​​​​​

ProbeProductExmax (nm)Emmax (nm)Cell stained
7-AAD​ab142391

488

647Dead
Calcein AMab141420495515Live
Nuclear Green DCS1ab138905503526Dead
Propidium Iodideab14083535617Dead
DRAQ7™​ab109202599/644678/697*Dead

*intercalated with DNA

Autofluorescence

​​​Naturally occurring cell components such as NADPH and flavins can emit fluorescence upon 488 laser excitation. Autofluorescence is influenced by cell type and physiological conditions. ​

To check if autofluorescence represents a problem in your experiment, an aliquot of unstained cells should be analyzed on the flow cytometer. Cell treatment and settings on the machine should be the same as the experimental sample. If autofluorescence is a problem, it can be dealt with by using a different laser, or mathematically corrected with compensation.

Compensate like you would do with any other fluorescence spillover, just treat the autofluorescence signal as an additional fluorochrome measured in an assigned detector and subtract it.

Refer to our fluorochrome chart or multi-color selector to find out if you can excite your dyes with a different laser.

Spectral overlap

When carrying out a multi-color flow cytometry experiment, the emission spectra of the various fluorochromes can overlap, resulting in detection in a different channel (also called spillover). This phenomenon – that can seriously affect your measurements – is controlled by compensation. Compensation is the process by which spectral overlap is estimated and subtracted from the total detected signal to yield an estimate of the actual amount of each dye. 

See our detailed guide to compensation for more information.


Fluorescence minus one (FMO)

As mentioned in the compensation section, fluorescent spectra can overlap and cause spillover. When running a multi-color flow cytometry experiment, the fluorescence minus one (FMO) control provides a measure of spillover in a given channel. This sample is stained with all the fluorescent conjugates except the one that is being tested, showing you the contribution of the other fluorescent conjugates in the signal of the unlabeled channel. The FMO control is fundamental in a multi-color experiment, because it allows for correct gating and select only the stained cells in the experimental sample, as shown in figure 1.

FMO

Figure 1. Schematic representation of a flow cytometry scatterplot after compensation in an experiment to discriminate CD4+ and CD4- T cells. The unstained gate and the FMO gate are shown. Note how the FMO gate allows better separation of positive from negative cells, compared to the negative control. (Adapted from Perfetto, S. P. et al., 2004)

NB. FMO provides a measure of spillover induced background, not of nonspecific antibody binding. Furthermore, it is not a measure of background staining that may be present when an antibody is included in that channel. To account for the former, refer to isotype control; for the latter, refer to negative controls and isoclonic controls.

Undesirable antibody binding

This broad term includes every instance of antibody binding that prevents correct interpretation of the data.

Negative controls

The negative control should be a population of cells that does not express the antigen of interest, use knockout cells if possible. This sample should be exposed to same experimental conditions as the population in study. Use this control for setting gating regions and discerning positive from negative cells.

​​​​​​Isotype controls 

Isotype controls are used to determine the background caused by nonspecific antibody binding. An isotype control uses an antibody of the same isotype as the primary antibody, but is specific for an antigen absent from the cells under study. Isotype controls should be used to determine the background due to nonspecific 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, fluorochrome type and number of fluorochrome molecules per immunoglobulin
  • Be derived by the same manufacturing process and presented in the same formulation

We offer a complete range of isotype controls, which are available both in unconjugated forms, and conjugated to Alexa Fluor® dyes, biotin, phycoerythrin, fluorescein isothocyanate (FITC), and other formats.

Featured isotype controls:

ProductDescriptionApplications
ab172730Rabbit monoclonal, IgGFlow cytometry, ICC/IF, IHC-P
ab171463Mouse monoclonal, IgG1
​(Alexa Fluor® 488)
Flow cytometry, ICC/IF, IHC-P
ab199093Rabbit IgG, monoclonal (Alexa Fluor® 647)Flow cytometry, ICC/IF

Refer to our complete guide to isotype controls for more information and products.

​​Isoclonic controls

The isoclonic control tells you whether the conjugate can mediate non-specific binding to the sample. Cells are stained with the conjugated antibody in an excess of identical (isoclonic) unlabelled antibody. In this control, the specific antibody binding sites are all taken up by the unconjugated antibody. The conjugated antibody can bind the sample only through the conjugate. If no fluorescent signal is detected, the conjugated antibody cannot bind non-specifically through its conjugate.

The isoclonic control informs whether the conjugate can mediate non-specific binding to the sample. Cells are stained with the conjugated antibody in an excess of identical (isoclonic) unlabelled antibody. In this control, the specific antibody binding sites are all taken up by the unconjugated antibody. The conjugated antibody can bind the sample only through the conjugate. If no fluorescent signal is detected, the conjugated antibody cannot bind non-specifically through its conjugate.

Like the isotype control, this is not a quantitative control, but solely qualitative.

Like the isotype control, this is not a quantitative control, but solely qualitative.




Head over to our ​flow cytometry protocols page for more practical advice.

References

Herzenberg, L. A., Tung, J., Moore, W. A., Herzenberg, L. A., & Parks, D. R. (2006). Interpreting flow cytometry data: a guide for the perplexed. Nature immunology, 7(7), 681-685.​​

Mahnke, Y. D., & Roederer, M. (2007). Optimizing a multicolor immunophenotyping assay. Clinics in laboratory medicine, 27(3), 469-485.

Hulspas, R., O'Gorman, M. R., Wood, B. L., Gratama, J. W., & Sutherland, D. R. (2009). Considerations for the control of background fluorescence in clinical flow cytometry. Cytometry Part B: Clinical Cytometry, 76(6), 355-364.

Nguyen, R., Perfetto, S., Mahnke, Y. D., Chattopadhyay, P., & Roederer, M. (2013). Quantifying spillover spreading for comparing instrument performance and aiding in multicolor panel design. Cytometry Part A, 83(3), 306-315.




Alexa Fluor® is a registered trademark of Life Technologies. Alexa Fluor® dye conjugates contain(s) technology licensed to Abcam by Life Technologies.