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High-Dimensional Immune Profiling of Human Retinal Detachment Samples Using Spectral Flow Cytometry: A Protocol for

Laura Molinero-Sicilia1,2,3, Alejandro G Del Hierro4, Nadia Galindo-Cabello1,2,3

  • 1Unit of Excellence Institute of Applied Ophthalmobiology (IOBA), University of Valladolid, 47011 Valladolid, Spain.

Methods and Protocols
|November 24, 2025
PubMed
Summary
This summary is machine-generated.

Retinal detachment triggers eye inflammation. This study presents a new method to analyze immune cells in the eye, aiding personalized treatments for retinal diseases and improving vision recovery.

Keywords:
gating strategyhigh-dimensional cytometryimmune markersintraocular immunophenotypingleukocyte profilingocular inflammationretinaretinal detachmentspectral flow cytometryvitreous

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Area of Science:

  • Ophthalmology
  • Immunology
  • Cell Biology

Background:

  • Retinal detachment (RD) compromises ocular immune privilege, leading to inflammation.
  • This inflammation can cause proliferative vitreoretinopathy and hinder visual recovery.
  • Understanding intraocular immune responses is crucial for developing targeted therapies.

Purpose of the Study:

  • To establish a standardized protocol for high-dimensional immune cell profiling in retinal detachment.
  • To enable detailed analysis of intraocular immune infiltrates using spectral cytometry.
  • To support the development of personalized immunomodulatory strategies for retinal diseases.

Main Methods:

  • Developed a protocol for collecting vitreous and retinal samples during RD surgery.
  • Integrated immune cell isolation (CD45+ sorting) with 39-color spectral cytometry.
  • Detailed sample preparation, staining, acquisition, and gating strategies for reproducible immunophenotyping.

Main Results:

  • The protocol allows for the identification of up to 62 distinct viable immune subsets per sample.
  • Functional status of immune cells can be assessed using 13 functional markers.
  • High-dimensional immune profiling is achievable from minimal biological material.

Conclusions:

  • The described protocol provides a robust platform for studying ocular inflammation in retinal detachment.
  • This method facilitates comprehensive analysis of immune cell landscapes in retinal diseases.
  • Enables mechanistic insights into immune responses, paving the way for novel therapeutic approaches.