In vitro binding and internalization assay using mouse NPC

Mouse NPC were prepared as described in a previous report4,13,14. Briefly, mouse livers were perfused with Liver Perfusion Medium (Gibco, 17701-038) to remove blood, and then perfused with a Collagenase solution (Wako, 034-22363) to digest the tissue. The cell suspension was centrifuged (50 × g, 4 °C, 3 min) three times to remove parenchymal cells. The supernatant was finally centrifuged (300 × g, 4 °C, 5 min), and the pellet was suspended in Endothelial cell medium (Sciencell, 1001).

To detect the cellular binding and uptake of the antibody, the Alexa Fluor 647-labeled anti-mouse FcγRIIB antibody was incubated with the mouse NPC for binding (4 °C, 60 min) and uptake (37 °C, 5 min). After the reaction, the cells were washed and stained with an anti-mouse CD146 antibody-FITC (Miltenyi, 130-102-230) and anti-mouse CD45 antibody-VioBlue (Miltenyi, 130-110-664). Finally, the fluorescence intensity of the samples was detected by BD FACSCanto II (Becton, Dickinson and Company, United States). In parallel with detecting the cellular signal, the fluorescence of the calibration beads (Bangs Laboratories, 647) was also measured. Cellular fluorescence was converted to the amount of antibody by using the calibration curve of standard beads and the labeling efficiency.

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