Biological Controls

Flow cytometry typically uses non calibrated arbitrary scale for fluorescence measurements. Reference samples (known negative and positive populations) are therefore needed for technical troubleshooting and for proper interpretation of the obtained data. These biological controls can be used to verify successful sample processing and staining, and also to help in gating.

Examples of negative control populations include:

  • Knock-out cell line or native cells (may also be naturally present [i.e. internal control] in heterogeneous samples, like PBMCs) previously proven not to express a specific marker. Comparison to unstained sample may reveal problems in staining due to unspecific binding. Useful in verifying assay specificity.
  • Wild type or mock-transfected cells are typically used as negative gating control when transfected cells are identified by the transgene expression (often a fluorescent protein, like GFP).

Examples of positive control populations include:

  • Cell lines or native cells with well-defined expression level(s) for the marker(s) of interest. Useful in verifying assay sensitivity and normalizing data between experimental runs.
  • Cells stimulated/treated with a standardized method to verify expected (strong) biological response in the samples. Useful as positive control to troubleshoot and normalize data in experimental procedures.

Examples of unspecified control populations include:

  • Unstimulated/untreated cells are typically used as negative gating control for cytokine and other assays where the basal marker expression level is non-negligible (or there is significant unspecific labeling), i.e. unstained or FMO control would underestimate the background.
  • Similarly, studies evaluating the effect of e.g. a specific disease or treatment/drug typically need a healthy or untreated control group as the meaningful comparison is not between negative and positive but rather between different expression levels in each group.

Unstimulated control

Fig. 1. Sometimes the “negative” population used to measure treatment response is not really negative but shows a measurable basal expression level of the marker of interest. Then unstained or FMO control is not suitable for gating purposes.

Additional Information:
  • Maecker and Trotter (2006) Flow cytometry controls, instrument setup, and the determination of positivity. Cytometry A. 69A:1037-1042, doi: 10.1002/cyto.a.20333