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A New Method for Lateral Visualization of the Primary Cilia on the Surfaces of Cells Cultured on White Glass Rods
Hidetoshi Tanioka1, Hideto Deguchi1, Shigeru Kinoshita2
1Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan.
Purpose:
To investigate the motility of the primary cilia of corneal endothelial cells (CECs), which exist like a hair on the cell surface, using our new in vitro method.
Methods:
A white glass rod was heated with a gas burner to produce a rod approximately 0.5 mm in diameter and 20 mm in length and then coated with collagen. A suspension of cultured human CECs (HCECs) was then added to the rod and cultured for 20 days. Cells on the rod's side were then observed using phase-contrast microscopy, and videos and images of the primary cilia were obtained. After fixing the cells cultured on the rod's surface, immunofluorescence staining was performed and fluorescence and phase contrast images were taken.
Results:
Hair-like structures were observed on the surface of live HCECs on the rod's surface. Video images revealed that the structures sometimes swayed owing to slight convection of the medium, yet had no motile function, and immunostaining with acetylated α-tubulin antibody confirmed that the structures were primary cilia.
Conclusions:
Our new method using white glass rods provided the ability to observe the movement of primary cilia in cultured living HCECs, and the findings clearly showed that the primary cilia of HCECs are passive rather than motile. This novel procedure can be applied widely to other cultured cells as a method to observe the movement of primary cilia from the lateral aspect of the cell.
Translational Relevance:
This method may help to clarify the role of primary cilia in the anterior chamber.
