μ slide 1 chambers

To study lymphocyte migration in the adhesion cascade within the hepatic sinusoids, cytokine-stimulated HSEC (TNF or TNF-α and IFN-γ for 24 h at 10 ng/ml) were grown to confluence in ibidi μ-slide VI chambers and connected to the flow system previously described (15 ). In some experiments IFN-γ stimulation was carried out for shorter periods (2h and 4h). For live cell imaging of lymphocyte migration we used a slightly modified flow-assay protocol to that used previously. Cytokine stimulated HSEC or HUVEC were seeded into μ-slide I chambers (Ibidi) were again prelabeled with CellTracker Green CMFDA (Thermo Fisher). The chambers were then connected to a Ibidi pump which allowed continuous perfusion of lymphocytes at a shear stress of 0.05Pa. Flow assays were performed with lymphocytes prelabeled with CellTracker Violet BMQC (Thermo Fisher) per the manufacturer’s guidelines. In some assays the endothelial cells were labeled with a Cell Mask™ plasma membrane stain as per the manufacturer’s guidelines. Some flow assays were performed with non-viable lymphocytes (cells underwent fixation with 4% paraformaldehyde). The endothelial monolayers and adherent lymphocytes were visualized and examined using a Zeiss 780 Zen microscope equipped with a 63 × 1.32 objective. Time lapsed confocal images and z-stacks were acquired and analyzed by Zen software.