Protocol for isolating and characterizing human vitreous immune cell infiltrates by flow cytometry and single-cell transcriptomic studies

Sandhya Rani Bhanja; Sayantan Ghosh; Pankaj Kumar; Tulasi Nagabandi; Arun Raina; Kaiser Alam; John V Forrester; Soumyava Basu

doi:10.1016/j.xpro.2025.103830

Protocol for isolating and characterizing human vitreous immune cell infiltrates by flow cytometry and single-cell transcriptomic studies

STAR Protoc. 2025 Jun 20;6(2):103830. doi: 10.1016/j.xpro.2025.103830. Epub 2025 May 17.

Authors

Sandhya Rani Bhanja¹, Sayantan Ghosh¹, Pankaj Kumar², Tulasi Nagabandi³, Arun Raina¹, Kaiser Alam¹, John V Forrester⁴, Soumyava Basu⁵

Affiliations

¹ Ocular Immunology Laboratory, Prof Brien Holden Eye Research Centre, LV Prasad Eye Institute, Kallam Anji Reddy Campus, Hyderabad, India.
² Strand Lifesciences, Bengaluru, India.
³ Center for Cellular and Molecular Biology, Hyderabad, India.
⁴ Ocular Immunology Group, Section of Infection and Immunity, Institute of Medical Sciences, University of Aberdeen, Aberdeen, UK.
⁵ Ocular Immunology Laboratory, Prof Brien Holden Eye Research Centre, LV Prasad Eye Institute, Kallam Anji Reddy Campus, Hyderabad, India; Saroja A Rao Center for Uveitis, LV Prasad Eye Institute, Kallam Anji Reddy Campus, Hyderabad, India. Electronic address: basu@lvpei.org.

Abstract

Vitreous infiltrating immune cells represent an accessible tissue sample that can provide valuable information on the intraocular immune response without compromising the structural and functional integrity of the eye. Here, we present our customized protocol for isolating and characterizing vitreous immune cell infiltrates by flow cytometry and single-cell transcriptomics. The protocol includes surgical modifications for sample collection and other measures to maximize the yield of immune cells from human vitreous samples. For complete details on the use and execution of this protocol, see Alam et al.¹.

Keywords: Cell Biology; Cell isolation; Flow Cytometry; Genomics; Health Sciences; Immunology; Molecular Biology; RNAseq; Sequencing; Single Cell.

MeSH terms

Flow Cytometry* / methods
Gene Expression Profiling* / methods
Humans
Single-Cell Analysis* / methods
Transcriptome* / genetics
Vitreous Body* / cytology
Vitreous Body* / immunology