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All tags Flow cyt Recommended controls for flow cytometry

Recommended controls for flow cytometry

Tips for choosing your standard flow cytometry experiment controls

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Along with the samples to be labeled, the following controls should be used whenever possible. The various positive controls are used for compensating and gating when setting up the flow cytometer.

ControlSample typePrimary
Secondary antibodyReason
Cells only - use treated and untreated cells

Negative control cells

NoNoNegative control/background autofluoresence control
Primary antibody control

Negative control cells

YesNoCheck for non-specific binding of primary
Treated primary antibody control

Treated cells

YesNoCheck for non-specific binding of primary antibody on treated cells
Isotype control

Negative control cells

Use isotype control antibody. This should be the same antibody isotype as primary antibody *YesTo confirm that the primary antibody binding is specific and not a result of non-specific Fc receptor binding or other protein interactions
Compensation controls for each fluorochromePositive population of labeled beads or positive control cell sampleYesYesPositive control to set up cytometer alignment and to remove spectral overlap
Cell viability control

Cell sample (identical to other samples) stained with both antibody and PI nuclear stain


Non-viable cells can be discriminated from live cells on the basis of light scatter (FSC=forward scatter)

This discrimination is often lost in fixed or permeabilized cells. In these cases dead cells can be distinguished from live cells by their uptake of fluorescent DNA dyes due to loss of membrane integrity

E.g. PI (propidium iodide) is used for deadcell discrimination in unfixed and non permeabilized cells. 7-AAD (7-aminoactinomycin D, fluorescent) + AD (actinmycin D, nonfluorescent) for fixed or permeabilized cells

Specificity control

Cell samples

Yes. With excess non-labeled primaryFor direct staining onlyAdd excess unlabeled primary antibody with normal amount of labeled primary. If staining is specific, the non-labeled primary should compete with labeled primary and reduce the fluorescence observed
Treated secondary antibody control

Treated cells


Check for non-specific binding of secondary antibody on treated cells

*Isotype controls can also be raised against an antigen known not to be present in the sample, e.g. KLH.