Controls in Flow Cytometry

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Controls are a vital part of any rigorous scientific process. Any experiment worth its peer-reviewed credentials requires controls to verify these data are valid, and that variables outside of the parameter of interest are not confounding the results in any way. In the rapidly-expanding field of flow cytometry, it is no different.  However, finding the appropriate controls can be a challenge at times depending on the flow cytometry assay.  Many factors can impact the results, and controlling for all of them requires no small amount of knowledge and finesse. Confounding factors range from spectral overlap of fluorophore channels, to cell autofluorescence impacting gating ease, to antibodies binding to off-target antigens or epitopes or a general lack of the commercially available controls1. At first glance, correcting and controlling for confounding variables in the flow cytometry process seems like a Herculean task. Luckily, there are some options for cytometric analysis out there that can help.

Gating controls

When interpreting data fresh off the cytometer, knowing where to place gates for certain surface antibody markers can be challenging at times. Most modern flow cytometry assays can test for multiple antibodies at one time, requiring multiple fluorescence channels to identify them.  Such an abundance of colors in one assay complicates analysis of the results immensely. While the premise of flow cytometry assumes that each fluorescent stain bound to a specific antibody will fluoresce only within a specific band of light, this is not the case. In reality, fluorophores emit some light outside of the target wavelength band, as well, which can affect the measurement of other antibodies in the affected bands. This is known as spectral overlap, and every assay must compensate for it in order to collect accurate results.  This method involves staining cells or commercially available compensation beads with only one antibody at a time and correcting for the spillover.


Once it is certain your assay compensation is correct there are different samples that can be collected within your experiment that can aide in setting the gates. One such way is through Fluorescence Minus One, or FMO, staining. Cells are stained with every antibody being measured except one. The gate then can be set on this negative staining to measure the fluorescence coming from the one target antibody. Another such sample is one using isotype controls.  These controls are typically the same class and type of primary antibody but lack specificity to the target. They can help differentiate non-specific binding from the specific antibody binding. Flow cytometry scientists are always having the debate on the use of FMO controls versus isotype control. Each can add value depending on the assay that is being performed. FMO controls can be expensive to process, especially with the more channels that are being evaluated. Also, with specimens where blood collection volumes can be limiting, such as animal models, it may not be possible to do a full combination of FMO controls. In these cases, isotype controls might be best. However, in some cases the isotype control may not be useful and gate placement might not be appropriate for your target of interest. This is especially seen in rare event populations. Back in 1999, Maurice R.G. O’Gorman and Joanne Thomas asked the question, “Isotype Controls – Time to Let Go?” In addition to their attempt to answer this question, this manuscript may be helpful when determining what is right for your assay2.

Assay controls

Control reagents (stabilized blood or lyophilized cells) with known, manufacturer-provided ranges are readily available from commercial sources such as Streck control reagents and Beckman-Coulter Cytotrol  and Immunotrol. However, these commercial control cells are often collected from normal donors and are not representative of most disease states.  In addition, these products are generally limited to defined ranges for basic immunophenotyping markers and lymphocyte subsets and often do not encompass a complex collection of markers characteristic of certain disease states.  Immortalized cell lines can be phenotypically characterized and used to mimic certain disease states as positive/negative controls in functional assays for cell therapeutics but the stability of the characteristics of these cell lines would need to be well understood.  In cases where biomarkers of interest can be induced/inhibited in normal samples, production of control cells can be achieved by artificial stimulation/inhibition with stocks of control cells produced and stabilized for longitudinal testing.


Commercial controls for intracellular markers such as cytokines are harder to find. Analysis of cell signaling with flow cytometry is a newer application of the technology when compared to surface antibody marker detection, and commercial products have yet to catch up. Fortunately, a few companies have developed a handful of new products that assist with intracellular analysis. Companies such as BD have HICK cells, which are good controls for cytokines such as IFNγ, TNFα and IL-2. Recently, Streck has promoted a new control called CD Chex Select, which offers a control for intracellular marker myeloperoxidate (MPO). This control offers reference ranges for other human surface markers, as well.  Recently, BioLegend, has also established a line of controls called Vericells. Not only do these evaluate common lymphocyte subsets but also can be used for intracellular markers. Again, depending on your assay would determine what type of control is appropriate.

As the field of flow cytometry grows and people think of new questions that can be explored with this technology, more and more products will be developed that will provide innovative solutions. What will be next?

Useful Information on Flow Cytometry Controls

Type of Control Description Comments

Set-up and daily use

Calibration Beads or use of stained samples

Check of optical alignment, fluorescence resolution intensity and instrument trend analyses

Fluorescence compensation controls

Use of hard dyed beads or using single stained samples, depending on the number of colors required

Hypothetically, there are several different combinations of fluorescence signals that need to be checked for compensation, but not all combinations can or need to be considered.  Certain instruments allow for compensation to be adjusted after acquisition

Fluorescence linearity

Monitoring linearity is a check of PMT voltages. A mixture of multi-level fluorescence beads or samples with known relative fluorescence intensity levels can be run

Acceptable mean fluorescence intensity (MFI) ranges for each type of bead in the mixture can be established by daily runs over a two week period. Levy-Jennings graphs can be utilized to plot the daily values and vendor service can be performed if a parameter falls outside of the expected range



Gating Controls



Fluorescence Minus One

Determination of the level of background fluorescence or auto-fluorescence for setting the negative gates.  Also ensure the observed staining is due to specific antibody binding to the target rather than an artifact. Useful in identifying a positive from a negative population especially when the expression levels are low

Binding Specificity

Blocking agents

Viability dyes

Non-specific binding can be common in flow cytometry assays and can lead to false positives.  Blocking agents can help ensure that only antigen specific binding is observed.


Live/dead staining can narrow gating to live cells, eliminating non specific binding associated with dead cells and debris. (Examples: Propidium iodide, TO-PRO-3 iodide, 7AAD, etc.)


Assay specific controls


Known negative samples such as characterized cell lines or cells where the antigen has been knocked down or the use of RNAi or CRISPR technologies to produce negative cells

Known positive samples may include characterized cell lines, commercial stabilized cell products with reference ranges or cells which have been transfected so the antigen is being over expressed to ensure positive staining

Unstimulated and fully stimulated samples may be important to determine both positive results and the dynamic range of fluorescence staining

Examples: BD© HICK cells, Streck© CD ChexSelect, Vericells, Streck CD Chex Plus, etc.


FCSL is a contract flow lab that provides high throughput and high capacity flow cytometry services, running multiple flow cytometers with up to 10 color antibody panels daily.  We are proficient in processing a multitude of specimen types including whole blood, frozen PBMCs along with cell culture and tissue processing capabilities. Our flexibility in handling so many specimen types allow for the support of a wide range of flow cytometry assays including: immunophenotyping/lymphocyte subset analysis, receptor occupancy, functional assays and cell viability/apoptosis measurements. Our expert staff is always available to help guide you through these tests and we welcome clients to visit our facility. We encourage sponsor engagement throughout the process. Contact us for more information!



1 Maecker HT, Trotter J.  2006. Flow cytometry controls, instrument setup, and the determination of positivity. Cytometry A. Sep 1;69(9):1037-42.

2 O’Gormann, M and Thomas, J.  1999.  Isotype Controls—Time to Let Go?  Cytometry (Communications in Clinical Cytometry) 38:78–80.