Protocol for measuring cilium length using 3D confocal fluorescence microscopy, CiliaQ software, and a quality control pipeline

Primary cilia are cellular antennas responding to various biological cues. Cilia undergo substantial changes in length, affecting their ability to sense extracellular signals and trigger downstream effector responses. Here, we present a protocol to image cilia in 2-dimensional cell culture using immunofluorescence staining and confocal microscopy. We describe steps to analyze cilium length using the CiliaQ plugin in Fiji/ImageJ and for using CiliaQ Explorer, a Python-based pipeline that plots CiliaQ-derived data and performs a statistical analysis after quality control. For complete details on the use and execution of this protocol, please refer to Hansen et al.¹.