To prepare the cells for flow cytometry analysis, a single-cell suspension containing 1 × 106 ADSC was prepared and subsequently fixed with 4% paraformaldehyde (Solarbio, Beijing, China). Following fixation, the cells were washed with PBS, and CD73 (PE; BD Biosciences, San Jose, CA, USA), CD90 (FITC; BD Biosciences), CD105 (APC; BD Biosciences), CD31 (APC; BD Biosciences), CD235a (PE; BD Biosciences), and CD45 (FITC; BD Biosciences) were used to label the cells. Flow cytometry was performed to determine the intensity of cell labelling.
Cd105 apc
Manufactured by BD
Sourced in United States, France
CD105-APC is a cell surface marker that binds to the endoglin protein. It is commonly used in flow cytometry applications to identify and characterize endothelial cells and their progenitors.
Automatically generated - may contain errors
Lab products found in correlation
Cd34 pe, by BD (12 mentions)
Cd90 fitc, by BD (9 mentions)
Cd73 pe, by BD (8 mentions)
Cd45 pe, by BD (5 mentions)
Cd45 fitc, by BD (5 mentions)
Cd90 pe, by BD (4 mentions)
Cd45 apc, by BD (4 mentions)
Hla dr fitc, by BD (4 mentions)
Cd44 fitc, by BD (3 mentions)
Cd29 apc, by BD (3 mentions)
Hla dr pe, by BD (3 mentions)
Cd73 fitc, by BD (2 mentions)
Cd45 apc h7, by BD (2 mentions)
Cellstack, by Corning (2 mentions)
Stemulate, by Cook Medical (2 mentions)
Cd73 v450, by BD (2 mentions)
Tryple, by Thermo Fisher Scientific (2 mentions)
Facsverse instrument, by BD (2 mentions)
Cd73 bv421, by BD (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
28 protocols using cd105 apc
1
Flow Cytometry Characterization of ADSCs
2
Isolation and Characterization of DPSCs
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Local ethical approval was granted by the Medical Ethics Committee, Faculty of Dentistry, Tongji University, Shanghai, China. hDPSCs were isolated from extracted premolars and third molars without caries or periodontal diseases. Informed consent was obtained from patients aged 18–25 years. Human umbilical vein endothelial cells (HUVECs) were obtained from the Chinese Academy of Sciences (category number: EAhy926). Rat DPSCs (rDPSCs) were isolated from the lower incisors of 3-week-old male Sprague-Dawley rats (weighing 90–110 g).
The “stemness” of the freshly isolated DPSCs was assessed by performing a flow cytometry analysis of the expression of the mesenchymal stem cell markers CD34-PE, CD105-APC (BD Bioscience), CD45-PE and CD90-PE (R&D Systems). Furthermore, the multilineage differentiation capacity of the DPSCs was confirmed by performing alizarin red S, alcian blue and oil red O staining to identify the osteogenic, chondrogenic, and adipogenic differentiation properties, respectively, using previously described methods.34 (link)
The “stemness” of the freshly isolated DPSCs was assessed by performing a flow cytometry analysis of the expression of the mesenchymal stem cell markers CD34-PE, CD105-APC (BD Bioscience), CD45-PE and CD90-PE (R&D Systems). Furthermore, the multilineage differentiation capacity of the DPSCs was confirmed by performing alizarin red S, alcian blue and oil red O staining to identify the osteogenic, chondrogenic, and adipogenic differentiation properties, respectively, using previously described methods.34 (link)
3
Proliferation and Immunophenotype of DPSCs
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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.
4
Immunophenotypic Characterization of BMSCs
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The BMSCs at passage 2 were digested with trypsin, washed with phosphate-buffered saline (PBS), and stained with CD29-APC (BD Biosciences, USA), CD44-FITC (BD Biosciences, USA), CD90-FITC (BD Biosciences, USA), CD105-APC (BD Biosciences, USA), CD34-PE (BD Biosciences, USA), CD45-PE (BD Biosciences, USA), and HLA-DR-PE (BD Biosciences, USA). The cells were washed and resuspended with PBS. Flow cytometry analysis was performed on the flow cytometer (BD Biosciences, USA).
5
Isolation and Characterization of hBM-MSCs
6
Characterizing Synovial MSC Phenotype
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Surface marker expression was analyzed by FACS Verse (BD) in synovial MSCs from four donors at passage 2. The cells before and 48 h after preservation were suspended in Hank’s balanced salt solution (HBSS) at a density of 5 × 105 cells/mL and stained for 30 min on ice with the antibodies CD44-PE-Cy7, CD73-V450, CD90-PE, CD105-APC, CD34-PE-Cy5, CD45-APC-H7, and CD31-FITC (all from BD), and Ghost Dye Violet 510 for dead cells (Tonbo Biosciences, CA, USA). These data were also analyzed using FlowJo software.
7
Characterization of hADSCs by Flow Cytometry
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Flow cytometry was employed to characterize hADSCs. Cells at passage 3 were digested with 0.25% EDTA-trypsin and washed thrice in PBS after centrifugation (1,500 × g for 5 min). 1 × 104 cells/tube were incubated in the dark at room temperature for 20 min with the following antibodies: CD44 FITC, CD105 APC, CD45 PE, CD34 PE (all purchased from BD Bioscience). Then, the cells were washed again twice with PBS, centrifuged (1,500 × g, 5 min) and resuspended in 200 μl PBS. Finally, CD surface antigens were analyzed with FlowCytometer (BD FACSCalibur, USA).
8
Cellular Viability and Apoptosis Assay
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For cellular viability assessment, the BM-MSC cells were washed with PBS twice, and then were trypsinized. The harvest cells were stained with CD90-FITC (BD) and CD105-APC (R&D system). In the case of co-culture assays the harvested MNC and CD34+ cells were stained with CD45-FITC (BD) and CD105-APC. In the case of MPN cell lines assays the harvested cells were previously labeled with CD90-FITC and CD45-APC, and then performed the annexin-V kit, as described below.
Cells from the different experimental conditions were stained with Annexin V-PE using the PE annexin V apoptosis detection kit (BD) following manufacturer's instructions. For cell cycle, the cells were stained with propidium iodite (PI), using the kit cycle tests (BD), according to manufacturer's recommendations. Samples were acquired on a FACSCalibur flow cytometer (BD) and analyzed using the Infinicyt software (Cytognos, Salamanca, Spain). Cell-cycle was analyzed using the ModFit LTTM Macintosh program (Verity Software, USA).
Cells from the different experimental conditions were stained with Annexin V-PE using the PE annexin V apoptosis detection kit (BD) following manufacturer's instructions. For cell cycle, the cells were stained with propidium iodite (PI), using the kit cycle tests (BD), according to manufacturer's recommendations. Samples were acquired on a FACSCalibur flow cytometer (BD) and analyzed using the Infinicyt software (Cytognos, Salamanca, Spain). Cell-cycle was analyzed using the ModFit LTTM Macintosh program (Verity Software, USA).
9
Immune Phenotyping of hDPSCs
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Immune-phenotypical characterization was performed by FACS analysis on immune-selected hDPSCs at passage 1. The expression of the typical mesenchymal stem cells (MSCs) markers, i.e., CD73, CD90, CD105, CD34, CD45, HLA-DR, was evaluated, as previously described by Pisciotta et al. [27 (link)]. Cells were stained with the following fluorochrome-conjugated antibodies (Abs): anti-human-CD73-PE-CY7, -CD90-FITC, -CD105-APC, -CD45-PE and -HLADR-PE-CY7 (all from BD Biosciences, Franklin Lakes, NJ, USA), and -CD34-ECD (Beckman Coulter, Fullerton, CA, USA). A minimum of 10,000 cells per sample was acquired and analyzed by using the Attune Acoustic Focusing Flow Cytometer (Attune NxT, Thermo Fisher, Waltham, MA, USA). Data was analyzed by FlowJo 9.5.7 (Treestar, Inc., Ashland, OR, USA) under MacOS 10.
10
Phenotyping of Thawed Synovial MSCs
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Human synovial MSCs were detached with TrypLE (Thermo Fisher Scientific) and suspended in FACS buffer (0.2% FBS and 5 mM EDTA [Thermo Fisher Scientific] in phosphate-buffered saline [PBS]) at a density of 5 × 105 cells/mL. Thawed MSCs were used immediately after thawing, with no further culture. The cells were stained for 30 min with the following antibodies: CD44 (PE-Cy7), CD45 (APC-H7), CD73 (V450), CD90 (PE), and CD105 (APC) (all from BD). Cell fluorescence was evaluated using a FACS Verse instrument (BD). The data were analyzed using FlowJo software (Tree Star Software, CA, USA).
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