Intracellular Staining Controls in Flow Cytometry

Author: Mike Blundell | Reviewer: Chloe Fenton

What Are Intracellular Staining Controls?

Intracellular staining controls are essential in flow cytometry to ensure accurate detection of intracellular targets such as cytokines, transcription factors, and signaling proteins. Unlike cell surface staining, intracellular staining requires fixation and permeabilization steps, which can make it more difficult to distinguish true signal from background, necessitating the use of appropriate controls.

Isotype controls have been optimized for cell surface staining to control for nonspecific binding of antibody and fluorophore. They are therefore only one of several controls that may be performed to accurately detect your positive population. Because intracellular staining requires fixation and permeabilization, which can affect antigen detection, autofluorescence, fluorophore brightness, and cell morphology, other controls are necessary. These can include biological controls, such as stimulation/blocking experiments and negative/positive controls, which are described on the Biological Controls page.

This page explains which intracellular staining controls to use, when to use them, and common mistakes to avoid when designing your flow cytometry experiments.

Which Intracellular Control Should You Use?

Common Mistakes with Intracellular Controls

• Relying on isotype controls alone for intracellular staining
• Ignoring effects of fixation on antigen detection
• Failing to include stimulated or blocked samples

Useful Intracellular Staining Resources

• Intracellular Staining Tips
• Intracellular Flow Cytometry — the good, the bad and the cytokines
• Intracellular Staining Protocols