Flow Cytometry Protocols

Volume 699 of the series Methods in Molecular Biology pp 165-178


Multiparameter Intracellular Cytokine Staining

  • Patricia LovelaceAffiliated withInstitute for Immunity, Transplantation, and Infection, Stanford University
  • , Holden T. MaeckerAffiliated withInstitute for Immunity, Transplantation, and Infection, Stanford University Email author 

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Intracellular cytokine staining (ICS) is a popular method for visualizing cellular responses, most often T-cell responses to antigenic or mitogenic stimulation. It can be coupled with staining for other functional markers, such as upregulation of CD107 or CD154, as well as phenotypic markers that define specific cellular subsets, e.g. effector and memory T-cell compartments. Recent advances in multicolor flow cytometry instrumentation and software have allowed the routine combination of 8–12 (or more) markers in combination, creating technical and analytical challenges along the way, and exposing a need for standardization in the field. Here, we will review best practices for antibody panel design and procedural variables for multicolor ICS, and present an optimized protocol with variations designed for use with specific markers and sample types.

Key words

Antigen specific Intracellular staining Multicolor Polychromatic Fixation Permea­bilization AIDS vaccine research T cells