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A Protocol for Harvesting Single-cell Suspension from Mouse Corneas.

Chao Huang1, Naikun Jiang1, Xueer Zheng1

  • 1Department of Ophthalmology, the Affiliated Hospital of Guizhou Medical University; School of Clinical Medicine, Guizhou Medical University.

Journal of Visualized Experiments : Jove
|April 20, 2026
PubMed
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Researchers developed a new protocol to create high-quality single-cell suspensions from mouse corneas. This method improves cell viability and purity, essential for advanced analyses like single-cell RNA sequencing.

Area of Science:

  • Ophthalmology
  • Cell Biology
  • Biotechnology

Background:

  • The cornea is vital for vision and ocular surface homeostasis.
  • Corneal cellular heterogeneity analysis is crucial for understanding eye diseases.
  • Preparing high-quality single-cell suspensions from dense corneal tissue is challenging.

Purpose of the Study:

  • To establish a robust protocol for generating single-cell suspensions from whole mouse corneas.
  • To ensure the generated cell suspensions are suitable for downstream applications like single-cell RNA sequencing (scRNA-seq).

Main Methods:

  • Mincing whole mouse corneas into fine fragments.
  • Sequential enzymatic digestion using collagenase A and trypsin.
  • Quality assessment via trypan blue staining, immunofluorescence, and RNA integrity analysis.

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Main Results:

  • The protocol consistently yields high cell numbers and excellent viability (≥ 94.6%).
  • Achieved a high proportion of single cells (96.3%).
  • Successfully identified major corneal cell types (Pancytokeratin, Keratocan) and verified RNA integrity.

Conclusions:

  • The developed protocol provides a reliable method for obtaining high-quality single-cell suspensions from mouse corneas.
  • This protocol meets the stringent requirements for advanced analyses, including scRNA-seq.
  • Facilitates deeper investigation into corneal biology and disease mechanisms.