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Broad Immune Phenotyping of Innate and Adaptive Leukocytes in the Brain, Spleen, and Bone Marrow of an Orthotopic Murine Glioblastoma Model by Mass Cytometry

Sophie A. Dusoswa, Jan Verhoeff, Juan J. Garcia‐Vallejo Cytometry PART A, Volume 95, Issue 4, 422-426 (2019)

PURPOSE: Here we present a 42 parameter panel to characterize myeloid immune cell subsets and T lymphocyte activation status in cryopreserved and barcoded single cell suspensions obtained from brain, spleen, and bone marrow of an orthotopic immunocompetent glioblastoma mouse model in a C57BL/6 background (Table 1). This panel is designed for mass cytometry by time of flight (CyTOF) and combines 34 antibodies against diverse cell surface and intracellular targets together with cisplatin for live/dead discrimination, iridium for cell identification, and six cellular barcodes 1 to enable simultaneous multiplexed acquisition of up to 20 samples (Table 2). The panel is designed for a comprehensive evaluation of the immune system in different organs during murine in vivo studies in the field of glioblastoma immunology, but could also be applied to other disease models in the central nervous system (CNS) with possible systemic involvement, such as brain metastasis arising from other types of cancer, experimental autoimmune encephalomyelitis (EAE), or neurodegenerative disease models. Marker selection was partly based on a combination of previously reported panels studying CNS immune infiltrates 2-7. The selected set of markers captures T lymphocytes (CD8+, CD4+, and regulatory T lymphocytes), dendritic cells (DC), monocytes, macrophages, microglia, tumor cells (when GFP positive), and granulocytes, and contains a set of antibodies to detect cell activation, migratory capacity and immune checkpoints (Table 2). The panel has been optimized with respect to marker selection, antibody clone usage, antibody‐metal pairing, and antibody concentration, and has room for additional markers by filling in a number of free channels as listed in Table 2.

CELL TYPE: Single cells from Miltenyi neural tissue dissociation kit (P) treated brain

MACHINE: Fluidigm CyTOF 3 Helios

Marker Target Clone Fluorochrome Purpose