Adipogenesis, chondrogenesis, and osteogenesis were tested with StemPro® kits (Gibco) which were used according to the manufacturer’s instructions (Additional file 1 ).
The immunophenotype of porcine WJCs was analyzed using mouse monoclonal antibodies to porcine CD31 (LCI-4):IgG1-RPE, CD45 (K252.1E4):IgG1-FITC, and SLA class II DR (2E9/13):IgG2b-FITC (AbD Serotec, Bio-Rad, Hercules, CA, USA), porcine CD105 (MEM-229):IgG2a-FITC and CD90 (5E10):IgG1-FITC (Abcam, Cambridge, MA, USA), and porcine CD44 (MEM-263):IgG1-FITC (Thermo Scientific, Middletown, VA, USA). All isotype control antibodies were derived from mice (IgG1-FITC, IgG1-RPE, IgG2a-FITC, and IgG2b-FITC) and were purchased from Invitrogen (Carlsbad, CA, USA). Antibodies for porcine CD73 were not available and monoclonal antibodies against human CD73 do not cross-react with porcine CD73 [10 (link), 11 (link)]. Cell suspensions (1 × 107 cells/ml in 100 μL PBS) were incubated with antibodies or isotype controls (final concentration 3 μg/mL) for 45 min at 4 °C in the dark. Fluorescence was analyzed with a microcapillary cytometer (Guava EasyCyte Plus and Cytosoft™ software, Millipore).
The immunophenotype of porcine WJCs was analyzed using mouse monoclonal antibodies to porcine CD31 (LCI-4):IgG1-RPE, CD45 (K252.1E4):IgG1-FITC, and SLA class II DR (2E9/13):IgG2b-FITC (AbD Serotec, Bio-Rad, Hercules, CA, USA), porcine CD105 (MEM-229):IgG2a-FITC and CD90 (5E10):IgG1-FITC (Abcam, Cambridge, MA, USA), and porcine CD44 (MEM-263):IgG1-FITC (Thermo Scientific, Middletown, VA, USA). All isotype control antibodies were derived from mice (IgG1-FITC, IgG1-RPE, IgG2a-FITC, and IgG2b-FITC) and were purchased from Invitrogen (Carlsbad, CA, USA). Antibodies for porcine CD73 were not available and monoclonal antibodies against human CD73 do not cross-react with porcine CD73 [10 (link), 11 (link)]. Cell suspensions (1 × 107 cells/ml in 100 μL PBS) were incubated with antibodies or isotype controls (final concentration 3 μg/mL) for 45 min at 4 °C in the dark. Fluorescence was analyzed with a microcapillary cytometer (Guava EasyCyte Plus and Cytosoft™ software, Millipore).
Free full text: Click here
