Detection of Intracellular Cytokines by Flow Cytometry

Abstract

Intracellular cytokine staining (ICCS), employing fluorescently labeled MAbs detected by flow cytometry, has emerged as the premier technique for studying cytokine expression at the single-cell level. Advances in polychromatic flow cytometry have dramatically enhanced the sophistication of ICCS investigations. ICCS can simultaneously measure multiple cytokines within a single cell, allowing the detection of complex cytokine phenotypes. Additionally, cytokines can be measured with a variety of other analytes, including transcription factors, proliferation dilution dyes, activation markers, and viability dyes. This capability, combined with the high throughput inherent in the instrumentation, gives ICCS an enormous advantage over other single-cell techniques such as ELISPOT, limiting dilution, and T cell cloning. The unit describes intracellular staining of cells that have already been stimulated in vitro and fixed. Methods for in vitro activation by PMA and ionomycin or antigens, fixation of cell suspensions, and cell surface staining are also described.

An optimized protocol for the generation and functional analysis of human mast cells from CD34 + enriched cell populations

“…Cells were then washed twice with 1 ml of PBS and resuspended in 250 μl of PBS. Cells were acquired on an LSR II flow cytometer and analyzed using FlowJo software (TreeStar, Inc, Ashton, OR)[10]. …”

“…Some activated cells were fixed with 4% paraformaldehyde. Intracellular cytokine staining was performed with PE-Cy7-IL-8 (Biolegend), Alexa fluor700-TNFα and Brilliant Violet 421-IL-6 (BD bioscience) antibodies [10]. These cells were then analyzed by flow cytometry.…”

Advances in Proteomic Techniques for Cytokine Analysis: Focus on Melanoma Research

“…Interestingly, the first cell sorter was introduced in the same year [ 183 ]. Flow cytometry is based on single-cell analysis, which allows to show the frequency of cytokine-expressing cells together with characterization of producing cell population using staining of intracellular cytokines together with cell markers [ 184 , 185 ] ( Figure 5 ). In the intracellular cytokine staining, cells are activated to produce cytokines and at the same time treated by substances that prevent cytokine secretion (such as Brefeldin A or monensin [ 186 ]).…”

High‐Dimensional Single‐Cell Analysis with Mass Cytometry

“…In order to see cytokine production, it is necessary to stimulate cells. In Basic Protocol 2, we outline a PBMC stimulation with PMA (protein kinase A activator) and ionomycin (ionophore; UNIT 6.24; Yin, Mitson-Salazar, and Prussin, 2015) to increase cytokine production in T cells. After the stimulation, cells are stained with cisplatin for identification of dead cells, and then surface stained.…”