Phenotypic Characterization of hAFSCs

Phenotypical characterization of hAFSCs was performed between passages 2 and 4, as previously described (Di Baldassarre et al., 2018b (link)). Briefly, cells were treated with the FIX & PERM^® Kit (Thermo Fisher Scientific) and then incubated for 1 h at RT with human anti-CD90-Alexa fluor 488-conjugated, CD34-Alexa fluor 488-conjugated, CD45-Alexa fluor 488-conjugated, SSEA4-Alexa fluor conjugated, OCT4-Alexa fluor 488-conjugated, Tra-1–60-Alexa fluor 488-conjugated, C-KIT-PE-conjugated, CD105-FITC-conjugated, NANOG-Alexa fluor 647-conjugated, SOX2-Alexa fluor 488-conjugated (All from Becton Dickinson, Franklin Lakes, NJ, United States) diluted 1:50–1:100 according to the manufacturer’s instructions. Cells incubated with isotypes (all from Becton Dickinson) were used as negative controls. Cytometric analysis was performed with a Cytoflex cytometer (Beckman Coulter Pasadenia, CA, United States), and data were analyzed with CytExpert Acquisition and Analysis Software (Beckman Coulter).