Goat sox2

AFSCs at first passage were sub-cultured until reaching 80% confluence. As previously reported [51 (link)], following trypsin dissociation, cells were stained with the following antibodies (mAbs): Rabbit-Thy-1 (CD90) and Rabbit-Endoglin (CD105) (Millipore, CA, USA), Mouse-SSEA4 (Cell Signaling Technology, MA, USA), Goat-Integrin β1 (CD29), Rabbit-HCAM (CD44) (Santa Cruz Biotechnology, CA, USA), Mouse-5’-Nucleotidase (CD73) (Gene Tex, CA, USA).
The expression of surface markers was analysed by indirect staining using secondary fluorochrome Alexa 488-conjugated antibodies (Abcam, Cambridge, UK). Non-specific fluorescence was assessed by using the secondary antibody alone. A minimum of 5,000 cells per sample was acquired and analysed using FACScan flow cytometer and Lysis II software (both from Becton Dickinson, San Jose, CA, USA).
The same cell samples were analyzed for nuclear stem cell markers, such as Goat- Sox2 (Santa Cruz Biotechnology, CA, USA) and Rabbit-Nanog and Rabbiti-Oct4, (Cell Signaling, MA, USA), after Fixation/Permeabilization process performed with the Staining Buffer Set (Miltenyi Biotec Inc, Auburn, CA, USA) optimized for nuclear staining.
After exposure to adipogenic differentiation medium, nuclear differentiation marker rabbit anti-PPAR (Santa Cruz, CA, USA) was analysed by using the Staining Buffer Set (Miltenyi Biotec Inc, Auburn, CA, USA).