Anti human cd146

Double immunofluorescent staining was performed to evaluate the phenotypic of CD140b⁺CD146⁺ cells. Some of the cells after magnetic microbeading against CD140b and CD146 were plated at low seeding density of 10–30 cells/cm² on fibronectin-coated 12-well plates and culture for 3–4 days. Cells were fixed with 4% paraformaldehyde for 20 min. Permeabilization was performed using 0.1% Triton-X 100 for 10 min and blocked with 2% BSA for 30 min. Cells were incubated with primary antibodies; anti-human CD140b (1:200; R&D Systems) and anti-human CD146 (1:100; Abcam, Cambridge, UK) antibodies at 4 °C overnight. The following day, cells were incubated with the secondary antibodies: donkey anti-mouse antibodies conjugated with Alexa Fluor 564 (1:200; ThermoFisher Scientific) and goat anti-rabbit antibodies conjugated with Alexa Fluor 488 (1:200; ThermoFisher Scientific). The cell nuclei were detected by DAPI (ThermoFisher Scientific). All washing steps were performed with PBS and conducted at room temperature unless specified. Images were captured using a Carl Zeiss LSM inverted confocal microscope and a Zeiss LSM Zen 2010 software (Carl Zeiss, Munich, Germany) at the Centre for PanorOmics Sciences (CPOS) Imaging and Flow Cytometry Core, The University Of Hong Kong.