Cd73 fitc

To investigate cellular proliferation, DPSCs were labelled with 5 μM of 5-chloromethylfluorescein diacetate (CMFDA) in α-MEM for 45 min at 37°C CO₂. Cells were washed in medium and seeded at 10⁵/well in a 12 well-plate, in duplicate, for different time points (2, 3, and 4 days). Cytoplasmic amount reduction of the dye was measured using NAVIOS flow cytometer (Beckman Coulter, Brea). The data were analysed with FlowJo software. For immunophenotype analysis, cells cultured for 1 week with 10% FBS or 1% PL were trypsinized and aliquoted in FACS tube. Cells were washed twice with PBS 0,1% BSA. To limit unspecific binding, a blocking step is performed by resuspension of the pellets with PBS 1% BSA for 15 min. Cells were stained on ice for 1 h with saturating concentrations of primary conjugated antibodies diluted 1 : 50 in PBS 0,1% BSA. CD13-PE (mouse IgG1), CD29-APC (mouse BALB/c IgG1), CD44-FITC (mouse IgG2b), CD45-APC-H7 (mouse IgG1), CD73-FITC (mouse IgG1), CD90-PE (mouse BALB/c IgG1), and CD105-APC (Mouse BALB/c IgG1) monoclonal antibodies purchased from BD (Franklin Lakes) and CD146-PE (mouse IgG1), CD34-FITC (mouse IgG2a), and HLA-DR-PE (recombinant human IgG1) monoclonal antibodies purchased from Miltenyi Biotec (Bergisch Gladbach) were used to define the MSC panel as previously described [15 (link)]. At least 10000 events were counted for each sample.

After cell detachment using a 0.125% trypsin solution, cells were washed with PBS and resuspended in PBS containing 2% FBS. Cell concentration and viability were monitored using Trypan blue in a Neubauer haemocytometer. The following monoclonal antibodies were used as indicated by the manufacturer (BD Pharmingen): CD90-PE (BD, #555596), CD73-FITC (BD, #561254), CD105-FITC (BD,#561443), CD45-FITC (BD,#347463), CD14-PE (BD,#555398), CD34-PEcy5 (BD,#561819), CD31-PE (BD,#555446), IgG-FITC (BD,#555786), HLA-DR-FITC (BD,#555558), CD166-PE (BD,#560903), CD44-PE (BD,#555479), CD54-PEcy5 (BD,#555512), CD146-PE (BD,# 559263). At least 20,000 events were acquired on a BD FACSCalibur flow cytometer and data was analyzed using CellQuest software.

BMMSCs were isolated, cultured and induced into osteoblast. The procedure was detailed in our previous studies [32 (link), 33 (link)]. The immunophenotype of BMMSCs were evaluated at P3 (the third passage). After 15 min incubation with the antibodies as follow: CD105-FITC, CD73-FITC, CD29-FITC, CD90-FITC, CD166-PE, CD34-PE, CD45-FITC, CD80-FITC, CD86-FITC (BD Biosciences Pharmingen, San Diego, California, USA), CD44-FITC (Miltenyi Biotec, Bergisch Gladbach, Germany), BMMSCs were analyzed by FACS Calibur system (BD, Mountain View, CA, USA) using Cell Quest software.

For identification analysis, hDPSCs were identified by the positive expression of MSC surface markers (CD73, CD90 and CD105) and the negative expression of hematopoietic antigens (CD34 and CD45). Human DPSCs at third passage were resuspended as single cell suspensions and incubated with fluorochrome-conjugated anti-human CD34-FITC, CD45-PE-CY5, CD73-FITC, CD90- PE-CY5, and CD105-PE (BD Biosciences, Franklin Lakes, NJ, USA) antibodies at 4 °C for 30 min. The samples were detected and analyzed by flow cytometry (BD Biosciences).

After cell detachment using a 0.125% trypsin solution, cells were washed with PBS and resuspended in PBS containing 2% FBS. Cell concentration and viability were monitored using Trypan blue in a Neubauer hemocytometer. The following monoclonal antibodies were used as indicated by the manufacturers (BD Pharmingen® (BD, Franklin Lakes, New Jersey, USA)): CD90-PE (BD, #555596), CD73-FITC (BD, #561254), CD105-FITC (BD, #561443), CD45-FITC (BD, #347463), CD14-PE (BD, #555398), CD34-PEcy5 (BD, #561819), CD31-PE (BD, #555446), IgG-FITC (BD, #555786), HLA-DR-FITC (BD, #555558), CD166-PE (BD, #560903), CD44-PE (BD, #555479), CD54-PEcy5 (BD, #555512), CD146-PE (BD, #559263). Isotype controls were used for determining nonspecific binding and defining cut-off values. A minimum of 20,000 events were acquired on a BD FACS Calibur® flow cytometer and results were analyzed using CellQuest™ software.