Quantifying NF‐κB Activation by Flow Cytometry of IκBα Degradation

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 IC 50 from flow cytometry data using Excel