Anti icam 1 antibody

C26 cells were labeled with 25 μM CFSE probe, (Thermo Scientific; MD, USA) by a 30 min incubation at 37 °C, followed by washing in the basal culture medium. Labeled cells were then resuspended to the experimental cell concentration of 2 × 10⁵ cells/ml. In some experiments, primary LSECs were incubated for 1 hour with the anti-ICAM-1 antibody (Thermo Fisher Scientific; MD, USA). In another set of experiments, LSECs freshly isolated from livers treated with ICAM-1 siRNA silencing or with an scramble siRNA were plated in basal media. Then, tumor cells were seeded onto the LSEC cultures. The resulting co-cultures were maintained for 30 min at 37 °C. Then, total emitted fluorescence was measured using Ascent Fluoroskan (Labsystems S.A.C.). Then, co-cultures were extensively but delicately washed with culture medium to prevent removing of adherent cells. The fluorescence emitted by adhered cells was again measured. Finally, the percentage of adhered cells was calculated by the subtraction of background fluorescence as follows: $$\%\mathrm{adhesion}=(\mathrm{fluorescence}\mathrm{emitted}\mathrm{by}\mathrm{adhered}\mathrm{C}26\mathrm{cells}\times 100)/\mathrm{total}\mathrm{fluorescence}$$

Cells were stained against E‐selectin by incubating the fixed TNF‐α stimulated cells with 50 μg/ml FITC labeled E‐selectin binding peptide, Esbp30 (purchased from GL Biochem, China, >95% purity validated by HPLC and MS),30 for 2 hr at room temperature. Also, ICAM‐1 receptors were stained using incubation with 2.5 μg/ml anti ICAM‐1 antibody (ThermoFisher, MA) for 2 hr, and labeled using a secondary antibody Alexa fluor 647 anti‐mouse (ThermoFisher, MA). Cell nuclei were stained using DAPI. The microfluidic channels were washed twice with PBS between staining steps. Confocal images were obtained using a Nikon based confocal microscopy system (Andor, Belfast GB) and recorded by an Andor DS‐Ri2 camera. Images were acquired by an Andor iQ3 software and fluorescent intensity analysis was performed via an ImageJ software. Data was averaged from 3 to 5 images per experiment and three repeat experiments were conducted for each condition.