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

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.