Igg1 fitc

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).

For phenotypical identification of AF-MSCs, at least 1 × 10⁵ cells for one assay were collected by centrifugation at 600 ×g for 5 min. Pelleted cells were washed twice in phosphate-buffered saline (PBS) supplemented with 0.2% foetal bovine serum (FBS) (Gibco, Life Technologies, Grand Island, NY, USA). Then cells were suspended in 50 μL PBS with 1% bovine serum albumin (BSA) and incubated with the following antibodies against cell surface markers: FITC conjugated mouse anti-human CD45 (BD Pharmingen, San Jose, CA, USA), CD34 (Miltenyi Biotec, Teterow, Germany), and CD90 (Molecular Probes, Life Technologies, Waltham, MA, USA) or PE labeled mouse anti-human CD105 (Invitrogen, Life Technologies, Waltham, MA, USA). Mouse IgG2A-FITC (Miltenyi Biotec, Teterow, Germany), IgG1-FITC (Invitrogen, Life Technologies, Waltham, MA, USA), or IgG1-PE (Molecular Probes, Life Technologies, Waltham, MA, USA) was used as isotype controls. Samples were incubated in the dark at 4°C for 30 min and washed twice with PBS with 1% BSA. Finally, cells were suspended in 200 μL PBS with 1% BSA and analyzed using Guava easyCyte 8HT flow cytometer (Millipore, USA) with InCyte 2.2.2 software.