Protocol for live-cell imaging and analysis of NF-κB localization and target gene transcription in Drosophila S2∗ cells

NF-κB transcription factor (TF) activation is heterogeneous across identically stimulated cells, shaping innate immune responses. Here, we present a protocol to quantify this heterogeneity in live Drosophila melanogaster S2∗ cells using dual reporters of NF-κB (Relish) subcellular localization and downstream transcription. We describe steps for reporter cell line generation, immune stimulation and time-lapse confocal imaging, and machine-learning-based single-cell analysis to classify signaling behaviors and predict transcriptional responses. For complete details on the use and execution of this protocol, please refer to Nawar et al. 1