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

Area of Science:

• Cell Biology
• Microscopy Techniques
• Bioinformatics

Background:

• Primary cilia act as cellular antennas, sensing extracellular signals.
• Cilia length is crucial for signal transduction and cellular responses.
• Changes in cilia length impact biological functions.

Purpose of the Study:

• To present a standardized protocol for imaging primary cilia in 2D cell cultures.
• To introduce computational tools for analyzing cilia length and associated data.
• To facilitate quantitative studies on cilia length dynamics.

Main Methods:

• Immunofluorescence staining of primary cilia.
• Confocal microscopy for high-resolution imaging.
• Utilizing the CiliaQ plugin in Fiji/ImageJ for length measurement.
• Employing CiliaQ Explorer (Python pipeline) for data analysis and statistics.

Main Results:

• A reproducible protocol for cilia imaging and length analysis is established.
• CiliaQ and CiliaQ Explorer enable efficient data processing and statistical assessment.
• The workflow allows for quality control and visualization of cilia length data.

Conclusions:

• The presented protocol and tools streamline the quantitative analysis of primary cilia length.
• This methodology supports research into how cilia length variations affect cellular functions.
• Accurate cilia length measurement is vital for understanding cilia-mediated signaling pathways.