Cd146 pe

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.

BD Stemflow™ Human MSC Analysis Kit (BD biosciences, California, USA), CD24-FITC, and CD146-PE (both bought from Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) were used on sorted CD105+ cells and were acquired using FACS Calibur for enumeration of CD24, CD146, CD73, CD90, CD105, CD11b, CD19, CD34, CD45, and HLA-DR markers. All antibody staining was prepared according to manufacturer protocol. Each sample was run with proper isotype control.

TSPCs (2 × 10⁵) were detached and stained for 20 min at 4 °C in the dark with fluorescently conjugated anti-human antibodies CD90-FITC (Clone 5E10, BioLegend, San Diego, CA, USA), CD105-PE (Clone SN6h, BioLegend), CD44-BV605 (Clone IM7, BioLegend), CD73-APC (Clone AD2, BioLegend), and CD146-PE (Clone 541-10B2, Miltenyi Biotec, Bergisch Gladbach, Germany) and analyzed using a CytoFLEX flow cytometer (Beckman Coulter Life Sciences, Brea, CA, USA). Collection of 50,000 events for each cell sample was performed. Subsequent gating strategies were standardized for each sample based on the following criteria: scatter, singlets, and positive expression. The resulting data were overlaid with corresponding isotype controls.

Flow cytometric analysis was performed on LD, HD, and LDHD using a CytoFLEX flow cytometer (Beckman Coulter Life Sciences). 2 × 10⁵ cells were suspended in staining buffer and incubated for 20 min at 4°C with fluorescently conjugated anti-human antibodies: CD90-FITC (Clone 5E10, BioLegend), CD105-PE (Clone SN6h, BioLegend), CD44-BV605 (Clone IM7, BioLegend), CD73-APC (Clone AD2, BioLegend), CD166-PerCP-eFluor™ 710 (Clone 3A6, Fisher Scientific), CD14-APC (Clone 61D3, eBioscience), CD45-VioBlue (Clone 5B1, Miltenyi), CD31-PE (Clone WM59, BD Biosciences), HLA-DR-PE-CF594 (Clone G46-6, BD Biosciences), CD10-APC (Clone HI10a, Biolegend), CD146-PE (Clone 541-10B2, Miltenyi Biotec), CD200-FITC (Clone OX104, Invitrogen), CD133-PE (Clone TMP4, eBioscience), and CD107-PerCP/Cy5.5 (Clone H4A3, BioLegend). Acquisition of 50,000 events for each cell sample was performed. Subsequent gating strategies were standardized for each sample based on scatter, singlets, and positive expression, which were overlaid with corresponding isotype controls.