View a pdf version of this page 
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 | Primary | Secondary | Reason |
|---|---|---|---|---|
Cells only | 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. |
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.
