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Quantifying NF-κB Activation by Flow Cytometry of IκBα Degradation
Praveen Krishna Veerasubramanian1,2, Bruce A Jacobson1,2, Fridrik J Karlsson1
1Inflammation and Immunology Research Unit, Pfizer, Cambridge, Massachusetts.
Insights
This study introduces a novel flow cytometry assay to measure inflammation by quantifying IκBα degradation, a marker for Nuclear factor-κB (NF-κB) activation in immune cells. This method offers a robust tool for immunology research.
Area of Science:
- Immunology
- Cellular Biology
- Molecular Biology
Background:
- Nuclear factor-κB (NF-κB) is a key pro-inflammatory transcription factor central to immune responses.
- Directly measuring active, nuclear NF-κB via flow cytometry is challenging due to antibody limitations.
- Assessing NF-κB activation is critical for understanding inflammatory processes and developing targeted therapies.
Purpose of the Study:
- To develop and validate a flow cytometry assay for quantifying NF-κB activation.
- To indirectly measure NF-κB activation by assessing the degradation of its inhibitor, IκBα.
- To provide a reproducible method for medium-to-high-throughput analysis of NF-κB activity in immune cells.
Main Methods:
- A flow cytometry protocol was established to quantify the intracellular levels of IκBα.
- The assay measures the decrease in IκBα as an indicator of NF-κB pathway activation.
- The protocol was validated using human primary regulatory T cells stimulated with TNFR2 agonists.
Main Results:
- The developed flow cytometry assay successfully detected IκBα degradation in response to inflammatory stimuli.
- The assay demonstrated utility in human primary regulatory T cells, T cells, and THP-1 cell-derived macrophages.
- The protocol provides a reliable method for assessing NF-κB activation across different immune cell types.
Conclusions:
- This protocol offers a robust and reproducible method for measuring NF-κB activation via IκBα degradation using flow cytometry.
- The assay is adaptable for various immune cells and inflammatory triggers, facilitating broader research applications.
- This technique enhances the ability to study inflammatory responses and screen potential immunomodulatory agents.
Abstract:
Nuclear factor-κB (NF-κB) is a crucial pro-inflammatory transcription factor whose activation is of immense interest to immunology research. Estimation of NF-κB activation through flow cytometry is not possible due to the unavailability of robust flow cytometry antibodies that can bind to its phosphorylated, active, nuclear form. In this protocol, we describe a flow cytometry assay that measures the activation of the pro-inflammatory transcription factor NF-κB in stimulated immune cells by quantifying the degradation of its upstream regulator IκBα. We demonstrate the utility of this protocol by assessment of intracellular IκBα in human primary regulatory T cells experiencing TNFR2 agonism, a process previously reported to activate NF-κB in these cells. We also show that this assay may be applied to study NF-κB activation in other cell types, such as human primary T cells and THP-1 cell-derived macrophages, when induced by their corresponding inflammatory cues. Thus, this robust and reproducible protocol will be of interest to a wide range of scientists who aim to measure NF-κB activity in medium-to-high-throughput assays. © 2024 Wiley Periodicals LLC. Basic Protocol: Quantifying inflammatory activation by flow cytometry of IκBα degradation Support Protocol 1: Isolating and expanding human regulatory T cells Support Protocol 2: Calculating IC50 from flow cytometry data using Excel.
