Pacific blue anti human cd31 antibody

Aliquots of 1 million isolated ECFCs from both donors were characterized by fluorescence activated cell sorting (FACS), and HUVECs were used as control. Briefly, cells were resuspended in cold 1% bovine serum albumin (BSA; pH = 7.0, Axonlab, in solution with PBS) and centrifuged at 300 g for 5 min at 4°C. The supernatant was discarded, and the cells again resuspended into 1% BSA/PBS at 4°C. 1% formaldehyde (FormaFix 4%, EBIS) was then added at the cells and the mixture was incubated for 10 min at 4°C. Cells were subsequently washed with 1% BSA/PBS and finally centrifuged. After resuspension with 1% BSA/PBS, the primary antibody (Pacific blue anti-human CD31 antibody, BioLegend, 2.0 µg per 1 M cells) was added and let incubated at 4°C for 30 min. Finally, ECFCs were again washed, centrifuged, and resuspended with 1% BSA/PBS. FACS was performed using High-end BD SORP flow cytometer (Fortessa, BD Biosciences) and BD FACSDiva software (BD Biosciences). Results were elaborated and analyzed with FlowJo software (FlowJo, LLC).

For FACS analysis, single cells derived from liver and organoids were suspended in DMEM plus 2% fetal bovine serum. Cell suspensions were analyzed using a BD FACSCalibur (BD Biosciences, San Jose, CA) or BD FACSAria II. For FACS, a BD FACSAria II cell sorter was used to isolate the target cell population. Single-cell suspensions of tumor cells were labeled with R-phycoerythrin (PE) anti-human CD140a antibody (PDGFRA, 5 μL per million cells in 100 μL volume, 323506; BioLegend, San Diego, CA), Pacific Blue anti-human CD31 antibody (2 μL per million cells in 100 μL volume, 102422; BioLegend), fluorescein isothiocyanate anti-human CD326 antibody (EpCAM, 5 μL per million cells in 100 μL volume, 324204; BioLegend), Alexa Fluor 700 anti-human CD45 antibody (1 μL per million cells in 100 μL volume, 135906; BioLegend), and PE anti-mouse CD140a antibody (PDGFRA, 5 μL per million cells in 100 μL volume, 135906; BioLegend). For cell sorting, PDGFRA+ for CAFs were collected and processed for RNA extraction and quantitative reverse-transcription PCR.

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.