Intracellular Cytokine Quantification
Intracellular Cytokine Quantification - Related Videos
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This video demonstrates the effect of tobacco product preparations, TPPs, on cytokine production from human peripheral blood mononuclear cells using flow cytometry. Treatment of the cells with TPP downregulates the production of pro-inflammatory cytokines, which is estimated after lipopolysaccharide stimulation, followed by immunostaining and flow cytometry analysis.
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Evaluation of Stromal Cytokine-Induced Intracellular Protein Phosphorylation Using Phospho-Flow Cytometry
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Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator
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Cellular ion transport can often be assessed by monitoring intracellular pH (pHi). Genetically Encoded pH-Indicators (GEpHIs) provide optical quantification of intracellular pH in intact cells. This protocol details the quantification of intracellular pH through cellular ex vivo live-imaging of Malpighian tubules of Drosophila melanogaster with pHerry, a pseudo-ratiometric genetically encoded pH-indicator.
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A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening
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Here, we describe a phenotypic assay applicable to the High-throughput/High-content screens of small-interfering synthetic RNA (siRNA), chemical compound, and Mycobacterium tuberculosis mutant libraries. This method relies on the detection of fluorescently labeled Mycobacterium tuberculosis within fluorescently labeled host cell using automated confocal microscopy.
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Assessing Antiviral Cytokine Production in Response to Viral Infection
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2026
Source: Zhang, J., et al. Dissecting Innate Immune Signaling in Viral Evasion of Cytokine Production. J. Vis. Exp. (2014)This video demonstrates the process of infecting murine fibroblast cells with herpesvirus and monitoring the resulting antiviral cytokine release and gene expression. It outlines key steps, including viral infection, media collection for cytokine quantification, and cell harvesting for gene expression analysis.
FAQs
Blocking protein secretion after stimulation allows cytokines to remain inside producing cells long enough for detection. This creates a measurable intracellular signal that can be assigned to individual cells, whereas secreted-cytokine assays report material released from the overall cell population. The distinction helps reveal functional immune subsets that bulk measurements may not distinguish.
Fixation and permeabilization prepare stimulated cells for antibody-based intracellular detection. Permeabilization creates access to cytokines located inside the cells, while the fixed preparation can then be stained with fluorophore-labeled antibodies. These steps are essential because surface-marker staining alone cannot reveal cytokine signals contained within the cellular compartment.
Flow cytometry measures the cytokine signal together with surface or intracellular markers in the same cells. Researchers can therefore associate cytokine production with defined immune populations rather than interpreting cytokine abundance alone. In cancer studies, this supports separate evaluation of cytokine-producing T cells, natural killer cells, and other immune subsets.
Secreted-cytokine assays can indicate what a stimulated cell mixture releases, but they may not identify which cells produced the signal. Intracellular cytokine quantification adds a functional, cell-resolved perspective by pairing cytokine detection with cellular markers. This comparison is useful when different immune populations may contribute differently to an overall cytokine measurement.
The workflow begins by stimulating the cells and then inhibiting protein secretion. Cells are subsequently fixed and permeabilized, stained with fluorophore-labeled antibodies, and analyzed by flow cytometry. Including surface or intracellular markers during staining allows the measured cytokine signal to be interpreted in relation to the identity of each immune population.
A study can combine intracellular cytokine signals with surface or intracellular markers and assess several parameters in the same analysis. This design helps characterize cytokine-producing T cells, natural killer cells, and other immune populations. The resulting profiles can be examined in relation to antitumor responses, immune suppression, or changes associated with treatment.
Multiparameter cytokine measurements can link functional immune activity to specific cell populations, producing profiles rather than a single bulk cytokine value. In cancer research, these profiles may help describe antitumor immune responses, identify patterns associated with immune suppression, and evaluate treatment effects. Such relationships provide a basis for investigating candidate biomarkers.