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Background: Inflammatory leukocytes in the ischemic brain modulate brain injury, but their functions are not fully understood. The purpose of this study is to establish a novel technique of mass cytometry (CyTOF) to identify the functions and roles of inflammatory cells in ischemic stroke mice, an alternative method to flow cytometry, without fluorescence cross contaminations.Methods: Leukocytes were isolated from the spleen, bone marrow, and ischemic brain of ischemic stroke mice. These cell samples were simultaneously stained with 16 metal-labeled immune cell phenotype markers, and then detected by CyTOF. Data were analyzed by Cytobank program.Results: By using the manual gating strategy, the results showed that stroke resulted in significant increases of microglia-derived macrophages (MiMΦs), monocyte-derived macrophages (MoMΦs), natural killer (NK), plasmacytoid dendritic cells (pDCs), conventional DCs (cDCs), B cells, CD4+ T, CD8+ T cells, γδ T, and regulatory T (Treg) cells (p<0.05). For the first time, we discovered that the number of Eff/mem CD4, and Eff/mem CD8 T cells was significantly increased in the ischemic brain (p<0.05), suggesting that these effector/memory T cells are important for brain injury. In addition, SAPDE analysis showed that individual leukocytes in the ischemic brain on day 3 after stroke had higher protein expressions of CD45, CD11b, CD11c, B220, CD3, CD4, CD8, CD44, and NK1.1 than those from sham brains, suggesting that these proteins may be actively involved in brain injury. As expected, the number of leukocytes was significantly reduced (p<0.05) in the spleen, an indicator of immunosuppression after stroke. Nevertheless, in the bone marrow, Treg were increased at day 1, neutrophils and CD8 T cells increased at day 3 after ischemia (p<0.05), while the number of the remaining subtypes was reduced.Conclusions: The novel technique of CyTOF is a powerful tool to quantify leukocyte cell types and protein expressions in individual cells. Our results suggest that effector/memory T cells are involved in brain injury, and upregulation of cell surface receptors in individual leukocytes may be important for their effects on brain injury.