Research Article

Systematic Study of the Immune Components after Ischemic Stroke Using CyTOF Techniques

Figure 1

Characterization of the brain immune cell populations using mouse MCAO model by CyTOF. (a) Experimental workflow: mice with stroke and sham surgery were euthanized for tissue collections. The ischemic brain hemisphere, blood, spleen, and bone marrow were collected at days 1, 3, 7, and 14 after stroke, and immune cells were isolated and barcoded by a combination of three palladium (Pd) mass tags. Cells from the same tissue types were collected at the same time points, pooled, and stained using metal-labeled 17 antibodies against cell surface markers. The CyTOF data was analyzed using Cytobank and presented by SPADE, ViSNE, and standard dot pot. The marker intensity was analyzed using a heat map. The cell number correlations were analyzed using R programming language and presented as network. (b) Representative gating method. Cell surface markers used for cell-type identification including CD45, CD11b, B220, NK1.1, CD3, CD8, CD4, CD44, CD25, and TCRβ. Illustrative SPADE tree (top), viSNE (bottom right), and standard dot pot (bottom left) to present identified immune cell types in the ischemic brain. In SPADE, the colored standards present the markers’ median expression levels, and the node sizes show the cell numbers. In viSNE, the color gradients show the markers’ intensity, and each dot represents a single cell. All of the figures are representative data on day 3 after stroke onset. (c) Illustrative SPADE identification of brain immune cells (day 3).