Flow cytometry protocol to quantify immune cells in the separated epithelium and stroma of herpes simplex...

Area of Science:

• Ophthalmology
• Immunology
• Virology

Background:

• Current flow cytometry methods analyze whole corneas, hindering precise immune cell localization.
• Distinguishing immune cell infiltration in the corneal epithelium versus stroma is crucial for understanding ocular inflammation.

Purpose of the Study:

• To develop and present a protocol for analyzing immune cells in separated corneal epithelium and stroma.
• To enable detailed investigation of immune responses in distinct corneal layers during herpes simplex virus-1 (HSV-1) infection.

Main Methods:

• Detailed protocol for herpes simplex virus-1 (HSV-1) infection in mouse models.
• Method for separating the corneal epithelium from the corneal stroma.
• Single-cell suspension preparation from isolated epithelial and stromal tissues.
• Flow cytometry assay for immune cell analysis in separated layers.

Main Results:

• Successful separation of corneal epithelium and stroma.
• Generation of single-cell suspensions from both layers.
• Enables distinct immune cell profiling in epithelial and stromal compartments.

Conclusions:

• The presented protocol allows for precise analysis of immune cells in the corneal epithelium and stroma.
• This method enhances the understanding of immune cell dynamics in ocular infections like HSV-1.
• Facilitates targeted research into localized immune responses within the cornea.