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. 2017 Aug 2;6(8):e006387. doi: 10.1161/JAHA.117.006387

Figure 2.

Figure 2

CCR5 maintains the adoptively transferred Tregs at the injured endothelium of the ischemic brain. In vivo 2‐photon images were acquired to observe transferred Tregs in the ischemic area at 1 day after transient middle cerebral artery occlusion (tMCAO). Cranial window and MCAO surgery were performed in the donor mice before imaging. Tregs were isolated from wild type (WT) or CCR5−/− donor mice and stained with CellTracker™ Deep Red Dye before injection (pseudocolored blue). Neutrophils/macrophages were labeled with intravenously injected rhodamine 6G (red). Blood vessels were labeled with intravenously injected fluorescent labeled dextran (green). A, Representative image taken from the cranial window (5 mm in diameter) that was created on the left parietal skull of the recipient mice. The MCA was observed in the middle of the coverslip. B, Representative 2‐photon images of Tregs (blue) and neutrophils or macrophages (red) in the brain vasculature (green). C, Quantification of Tregs frequency in the video recorded over 200 seconds. D, Quantification of Treg migration speed. E through L, Kymographs of Treg‐endothelial interaction and Treg‐neutrophil/macrophage interaction were both generated based on the cell contact time between those cells in videos taken over 30 seconds. E, Representative 2‐photon images and corresponding kymograph of a single Treg (blue, white arrow) interacting with the endothelium (green). F, Kymograph of Treg‐endothelium interactions with multiple Tregs from 6 individual videos of each group. G, Pooled cumulative Treg‐endothelium contact time over the 30 seconds from 6 videos/group. H, Contact time of each Treg interacting with endothelium identified in the videos. I, Representative 2‐photon images and corresponding kymograph of a single Treg (blue) interacting with neutrophils/macrophages (red). J, Kymograph of Treg‐neutrophil/macrophage interactions with multiple Tregs from 6 individual videos of each group. K, Pooled cumulative Treg‐neutrophil/macrophage contact time over 30 seconds from 6 videos/group. L, Contact time of each Treg interacting with neutrophils/macrophages identified in the videos. **P≤0.01, ***P≤0.001. CCR5 indicates C‐C chemokine receptor type 5; Treg, regulatory T cell.