Canine Adipose-Derived Stem Cell Phenotype

Phenotype analysis was performed as described previously [77 (link)]. Passage 2 cADSC were placed in fluorescence-activated cell sorting (FACS) tubes (BD Biosciences, Franklin Lake, NJ, USA; 2 × 10⁵ cells/tube), washed with FACS buffer (PBS containing 2% FBS), followed by blocking Fc receptors with canine Fc receptor binding inhibitor (Thermo Fisher Scientific), and then incubated on ice for 20 min with the following fluorescein (FITC)- or phycoerythrin (PE)-conjugated antibodies: anti-CD14-FITC (BD Pharmingen, San Diego, CA, USA), anti-CD29-PE (BioLegend, San Diego, CA, USA), anti-CD34-PE (R&D Systems, Minneapolis, MN, USA), anti-CD44-PE (BioLegend), anti-CD45-FITC (eBioscience, San Diego, CA, USA), and anti-CD90-PE (eBioscience) or their respective isotype controls. The cells were washed twice with FACS buffer and resuspended in 500 μL FACS buffer. Fluorescence was evaluated by flow cytometry in a CytoFLEX instrument (Beckman Coulter, Brea, CA, USA). The data were analyzed using CytExpert ver2.0 analysis software.