Biological Controls in Flow Cytometry

Author: Mike Blundell | Reviewer: Chloe Fenton

What Are Biological Controls in Flow Cytometry?

Biological controls in flow cytometry are experimental samples used to confirm that the observed staining reflects true biological expression rather than technical artifact. These controls include known positive and negative samples, as well as stimulated or inhibited conditions, which allow researchers to validate staining specificity and interpret results accurately.

Unlike reagent-based controls, biological controls assess functional responses and changes in antigen expression. They are essential for confirming that detected signals represent real biological effects, particularly in complex assays such as intracellular staining or cytokine analysis.

This page explains when to use biological controls, the different types available, and how they improve the reliability of flow cytometry data.

Types of Biological Controls

Common types of biological controls include:

• Cells known from the literature to either express or lack expression of the antigen of interest
• Cells where the antigen has been knocked down or out, using RNAi or CRISPR technology to produce a negative cell
• Cells that have been transfected and the antigen is being overexpressed to ensure positive staining

Stimulated and Unstimulated Controls

In experiments such as cytokine release or signaling studies, unstimulated and fully stimulated samples are used to define both negative and positive responses, as well as the dynamic range of fluorescence staining.

When Should You Use Biological Controls?

Biological controls should be included when validating whether staining reflects true biological expression or functional response. They are particularly important when assessing cytokine production, signaling pathways, or changes in antigen expression under experimental conditions.

• When confirming whether a marker is truly expressed or absent
• When measuring functional responses such as stimulation or inhibition
• When validating intracellular staining results
• When comparing treated versus untreated or disease versus control samples

How Biological Controls Compare to Other Flow Cytometry Controls

Different types of controls in flow cytometry provide complementary information. The table below shows how biological controls compare to other commonly used controls.