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

## TL;DR

This paper provides a detailed protocol for measuring cilium length using 3D confocal imaging and a custom software pipeline.

## Contribution

The novel contribution is a complete workflow combining 3D imaging, CiliaQ software, and a Python-based analysis pipeline for accurate cilium length measurement.

## Key findings

- A protocol for immunolabeling and 3D confocal imaging of cilia in cell culture is described.
- CiliaQ plugin and Python pipeline enable segmentation, quantification, and statistical analysis of cilium morphology.
- The method is applicable to various ciliated systems beyond cell culture, including tissues and organoids.

## Abstract

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

•Protocol for immunolabeling and 3D confocal imaging of cilia in cell culture•Procedure to segment and quantify cilia morphology in 3D via the ImageJ plugin CiliaQ•Python pipeline to analyze CiliaQ data (plotting, statistics, and quality control)•Applicable to other ciliated systems, including tissue and organoids

Protocol for immunolabeling and 3D confocal imaging of cilia in cell culture

Procedure to segment and quantify cilia morphology in 3D via the ImageJ plugin CiliaQ

Python pipeline to analyze CiliaQ data (plotting, statistics, and quality control)

Applicable to other ciliated systems, including tissue and organoids

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

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.