Fig. 1

eCIRP increases neutrophil trogocytosis during TEM. Bone marrow neutrophils (BMNs) were stained with CellTrace Violet (blue pseudo color used for illustration) and mouse lung vascular endothelial cells (MLVEC) were stained with green-fluorescent lipophilic membrane staining dye, PKH67, immediately before the assay. A The MLVECs were cultured in the transwell insert for 2 days prior to experiment. Then freshly prepared BMNs were added to the insert with either PBS or eCIRP (0.5 µg/mL). The chemoattractant, fMLP (1 nM), was added to the bottom chamber. A Montage of z-stack images of the trans-endothelial migrated neutrophils generated with fiji ImageJ. The confocal images were taken by Zeiss LSM880. The arrow indicates the piece of the endothelial cell membrane carried by neutrophils. Scale bar, 10 µm. B An enlarged image of a single slice of the z-stack images in B (red box) shows green-fluorescent punctum sitting on the blue stained neutrophil. Scale bar, 10 µm. C Percent PE positive (+ve) trans-endothelial migrated BMN (TBMN) were quantified by confocal microscopy. D Confocal microscopic 3-dimensional reconstruction image of z-stack showing cells migrating through the trans-well membrane in the presence of eCIRP (0.5 µg/mL) at 2 h after treatment. The arrows indicate the endothelial membrane piece carried by the migrated neutrophils. Scale bar, 10 µm. Data represented as mean ± SEM. *p < 0.05, unpaired, two-tailed student’s t-test