High-throughput Imaging and Analysis Workflow for Evaluating Skin Cell Phenotypes and Proliferation States in Tissue...

Area of Science:

• Regenerative medicine
• Tissue engineering
• Cell biology

Background:

• Skin regeneration involves complex cellular interactions, with peripheral glial cells crucial for repair.
• Current technological limitations hinder a complete understanding of these regenerative processes.
• Accurate identification of proliferating cells is essential for studying tissue repair.

Purpose of the Study:

• To characterize cellular composition changes during skin regeneration.
• To overcome limitations in detecting dividing cells in skin tissue.
• To develop an advanced imaging and analysis protocol for high-resolution tissue studies.

Main Methods:

• Iterative-bleaching-extends-multiplexity (IBEX) method combined with Click-iT EdU chemistry.
• High-throughput imaging using a fully automated spinning disk confocal microscope.
• Development of an open-source image processing pipeline for data analysis.

Main Results:

• The adapted IBEX method accurately identifies and categorizes actively dividing cell types in intact skin.
• The combined approach overcomes underestimation of cell proliferation by traditional markers.
• A novel, user-friendly image processing pipeline facilitates analysis of highly multiplexed imaging data.

Conclusions:

• The IBEX protocol is versatile and adaptable to conventional imaging platforms for studying tissue regeneration.
• Combining IBEX with Click-iT EdU labeling provides a powerful tool for dissecting dynamic cellular processes in skin.
• This protocol advances the study of skin repair by enabling detailed spatial and temporal analysis of cell proliferation.