Cilia, 1st ed. 2024 Methods and Protocols Methods in Molecular Biology Series, Vol. 2725

This volume covers the latest advancements in the study of ciliary complexity. Protocols cover genomic, proteomic, imaging, and functional analysis of different ciliated tissues and their wide applicability in cilia biology. Chapters in this book primarily focus on methods to study multiciliated cells, and discuss topics such as SARS-CoV-2 infections of human primary nasal multiciliated epithelial cells; expansion microscopy of ciliary proteins; live-imaging centriole amplification in mouse brain multiciliated cells; biophysical properties of cilia motility; and mucociliary transport device construction. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Cutting-edge and thorough, Cilia: Methods and Protocols is a valuable resource for researchers who are interested in learning more about this developing field. 

Preface…
Table of Contents…
Contributing Authors… 

1. Combination of CRISPR-Cas9-RNP and Single-Cell RNAseq to Identify Cell State-Specific FOXJ1 Functions in the Human Airway Epithelium
Laure-Emmanuelle Zaragosi, Alizé Gouleau, Margot Delin, Kevin Lebrigand, Marei-Jeanne Arguel, Cedric Girard-Riboulleau, Geraldine Rios, Elisa Redman, Magali Plaisant, Rainer Waldmann, Virginie Magnone, Brice Marcet, Pascal Barbry, and Gilles Ponzio

2. SARS-CoV-2 Infection of Human Primary Nasal Multiciliated Epithelial Cells Grown on Air-Liquid Interface Cultures
Maria Victoria Humbert, Christopher J. McCormick, and Cosma Mirella Spalluto

3. A Chemically Inducible Organelle Rerouting Assay to Probe Primary Cilium Assembly, Maintenance, and Disassembly in Cultured Cells
F. Basak Turan, M. Erdem Ercan, and Elif Nur Firat-Karalar

4. Expansion Microscopy of Ciliary Proteins
Sohyeon Park and Xiaoyu Shi

5. Immunolabel-First-Expand-Later Expansion Microscopy Approach using Stable STED Dyes 
Dong Kong, Delgermaa Luvsanjav, and Jadranka Loncarek 

6. Structural Analysis of Sperm Centrioles Using N-STORM
Abigail Royfman, Sushil Khanal, and Tomer Avidor-Reiss

7. A Novel Sandwich Method for Serial Block Face SEM Imaging of Airway Multiciliated Epithelium
Nobuhiro Morone, Maria Guerra Martin, Patricia Goggin, Kirk Czymmek, Vito Mennella, and Jaime Llodra Gonzalez

8. Airway Cells 3D Reconstruction via Manual and Machine-Learning Aided Segmentation of Volume EM Datasets 
Aaran Vijayakumaran, Analle Abuammar, Odara Medagedara, Kedar Narayan, and Vito Mennella

9. Endogenous Tagging of Ciliary Genes in Human RPE1 Cells for Live-Cell Imaging
Stefanie Kuhns, Alice Dupont Juhl, Zeinab Anvarian, Daniel Wüstner, Lotte B. Pedersen, and Jens S. Andersen

10. Live-Imaging Centriole Amplification in Mouse Brain Multiciliated Cells
Amélie-Rose Boudjema, Adel Al Jord, Anne-Iris Lemaître, Marion Faucourt, Nathalie Delgehyr, Nathalie Spassky, and Alice Meunier

11. Proximity Mapping of Ciliary Proteins by BioID
Melissa Iazzi, Jonathan St-Germain, Saujanya Acharya, Brian Raught, and Gagan D. Gupta

12. Affinity Purification of Intraflagellar Transport (IFT) Proteins in Mice using Endogenous Streptavidin/FLAG Tags
Tina Beyer, Tiago Martins, Jeshmi Jeyabalan Srikaran, Marian Seda, Emma Peskett, Franziska Klose, Katrin Junger, Philip L. Beales, Marius Ueffing, Karsten Boldt, and Dagan Jenkins

13. Primary Human Nasal Epithelial Cell Culture
Mystica Terrance, Tarini Gunawardena, Hong Ouyang, Julie Avolio, Wenming Duan, Sowmya Thanikachalam, and Theo J. Moraes

14. BMI1 Transduction of Human Airway Epithelial Cells for Expansion of Proliferation and Differentiation 
Ruhina Maeshima, Amy Jacobs, Melis T. Dalbay, and Stephen L. Hart

15. High Speed Video Microscopy of Ependymal Cilia in Brain Organotypic and Cell Culture Models
William J. Dawes, Oriane Grant, Sam C. Reitemeier, Sarah Tetlow, Dani Lee, Robert A. Hirst, and Christopher O’Callaghan

16. Measuring Biophysical Properties of Cilia Motility from Mammalian Tissues via Quantitative Video Analysis Methods
Erika Causa, Ricardo Fradique, and Pietro Cicuta

17. Mucociliary Transport Device Construction and Application to Study Mucociliary Clearance
Patrick R. Sears and Lawrence E. Ostrowski

Subject Index List…

Includes cutting-edge methods and protocols

Provides step-by-step detail essential for reproducible results

Contains key notes and implementation advice from the experts