Flow cytometry - recommended controls

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.

Control

Sample
type

Primary
ab

Secondary
ab

Reason

Cells only
Use treated and untreated cells

Neg control cells

No

No

Negative control/background autofluoresence control

Primary ab control

Neg control cells

Yes

No

Check for non specific binding of primary

Treated primary ab control

Treated cells

Yes

No

Check for non specific binding of primary ab on treated cells

Isotype control

Neg control cells

Use isotype control antibody.This should be the same antibody isotype as primary antibody. *

Yes

To confirm that the primary antibody binding is specific and not a results of non-specific Fc receptor binding or other protein interactions.

Compensation controls for each fluorochrome

Positive population of labeled beads or positive control cell sample

Yes

Yes

Positive 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

Yes

Yes

Nonviable 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 primary.

For direct staining only

Add 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 ab control

Treated cells

No

Yes

Check for non specific binding of secondary ab on treated cells.

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