Isolation and Flow Cytometric Analysis of Natural Killer Cells from Human Glioblastoma Multiforme (GBM) Tissues

Abstract We present a reproducible protocol for isolating and analyzing natural killer (NK) cells from freshly resected human glioblastoma multiforme (GBM) tissue using flow cytometry. The workflow yields viable single‐cell suspensions suitable for downstream applications, including cell sorting, immunophenotyping, functional assays, and single‐cell RNA‐seq.© 2025 Wiley Periodicals LLC. Basic Protocol 1 : Dissociation of glioblastoma multiforme tissue to single‐cell suspension Support Protocol 1 : Preparation of digestion dissociation mix for dissociation of glioblastoma multiforme tissue Alternate Protocol 1 : Mechanical‐only dissociation of glioblastoma multiforme tissue (no‐enzyme control) Basic Protocol 2 : Mononuclear cell enrichment and red blood cell lysis of glioblastoma multiforme single‐cell suspension Support Protocol 2 : Removal of cellular debris from glioblastoma multiforme single‐cell suspension Basic Protocol 3 : Flow cytometry staining and acquisition for identification of tumor‐infiltrating NK cells from glioblastoma multiforme tissue Alternate Protocol 2 : Panel splitting for flow cytometry using instruments with limited numbers of channels