Monkey NPC was purchased from Ina Research, Inc. Briefly, livers were perfused with Hanks-HEPES buffer and a Collagenase solution. The cell suspension was centrifuged (50 × g, 4 °C, 1 min) twice, and finally centrifuged (1000 × g, 4 °C, 5 min) to give NPC. The NPC cells were then purified by density-gradient centrifuge, and suspended in Hepatocyte culture medium (Lonza, CC-3198).
To detect the cellular binding and uptake of the antibody in monkey NPC, the Alexa Fluor 488-labeled anti-monkey FcγRIIB antibody (Sino Biological, 90014-R046) was incubated with the monkey NPC to allow binding (4 °C, 60 min) and uptake (37 °C, 10 min) to occur. After the reaction, cells were washed and stained with anti-human CD31 antibody-Pacific Blue (BioLegend, 303114) and anti-non human primate CD45 antibody-APC (Miltenyi, 130-091-900). Finally, the fluorescence intensity of the samples was detected by BD FACSCanto II (Becton, Dickinson and Company, United States). In parallel with detecting cellular signal, the fluorescence of the calibration beads (Bangs Laboratories, 488) was detected.
To detect the cellular binding and uptake of the antibody in monkey NPC, the Alexa Fluor 488-labeled anti-monkey FcγRIIB antibody (Sino Biological, 90014-R046) was incubated with the monkey NPC to allow binding (4 °C, 60 min) and uptake (37 °C, 10 min) to occur. After the reaction, cells were washed and stained with anti-human CD31 antibody-Pacific Blue (BioLegend, 303114) and anti-non human primate CD45 antibody-APC (Miltenyi, 130-091-900). Finally, the fluorescence intensity of the samples was detected by BD FACSCanto II (Becton, Dickinson and Company, United States). In parallel with detecting cellular signal, the fluorescence of the calibration beads (Bangs Laboratories, 488) was detected.