Cd54 fitc

Phenotypical characterization of ASCs was performed using the BD FACSCalibur and analyzed with the CellQuest software (v. 1.0.1, Becton–Dickinson, Heidelberg, Germany). The cells were trypsinized, placed on ice for 30 min, and treated with the following labeled stemness-associated antigen markers: CD31-, CD34+, CD45–, CD54–, CD90+, CD105+, CD166+, HLA-ABC+, HLA-DR–. CD34-PE, CD90–FITC, and CD105-PerCP were part of the BD Stemflow Kit (Becton Dickinson, 562245). The other antibodies with the indicated specifications were purchased separately: CD31-FITC (R & D, Systems, Wiesbaden, Germany, FAB3567F), HLA-DR-PE (Becton Dickinson, 347401), CD166-PE (Becton Dickinson, 559263), CD34-APC (Becton Dickinson, 555824), CD54-FITC (Beckman Coulter, Krefeld, Germany, PN IM0726U), HLA-ABC-FITC (Becton Dickinson, 555552), and CD45-PE (Becton Dickinson, 555483). Table 1 shows the results from three donors. All experiments shown in this paper were performed with cells until reaching passage 5.

Analysis of cluster differentiation markers was achieved by flow cytometry with a Gallios analytical flow cytometer (Beckman Coulter, Irving, TX, USA). Aliquots (100 µL) of peripheral blood were mixed with the following fluorochrome-conjugated antibodies: Anti-human CD3-APC, CD4-APCeF750, CD8-PC7, CD14-PC5.5, CD16-PB, CD45-KrO, CD54-FITC, and CD56-PE (Beckman Coulter, Irving, TX, USA), and lysed with the Beckman Coulter Versalyse solution following the manufacturer’s recommendations. To obtain the cell counts for the different populations 100 µL of Flow-Count Fluorospheres (Beckman Coulter, Irving, TX, USA) were added.