Both fresh and thawed hUC-MSCs at passage 4 were used to determine the expression level of surface markers by flow cytometry. To perform phenotype characterization, the cells were incubated with antibodies labeled with fluorochromes as follows: CD90-PE, CD73-PE, CD105-PE, CD34-FITC, CD45-FITC, and HLA-DR-FITC (all antibodies purchased from BD Biosciences). The specific method was as follows: each primary antibody was added per 1 × 106 cells separately and incubated at room temperature for 1 h. Isotype-matched antibodies were used as controls. Then, the cells were washed again with PBS and analyzed by flow cytometry (FACS Calibur C6, BD).
Facscalibur c6
Manufactured by BD
Sourced in United States
The FACSCalibur C6 is a flow cytometry system designed for cell analysis and sorting. It features four-color detection capabilities and is equipped with two lasers to provide multiple parameters for comprehensive cellular analysis.
Automatically generated - may contain errors
Lab products found in correlation
Microsoft excel 2013, by GraphPad (2 mentions)
Prism 7, by GraphPad (2 mentions)
Cd90 pe, by BD (1 mentions)
Cd34 fitc, by BD (1 mentions)
Cd105 pe, by BD (1 mentions)
Cd45 fitc, by BD (1 mentions)
Cd73 pe, by BD (1 mentions)
Hla dr fitc, by BD (1 mentions)
Cisplatin, by Merck Group (1 mentions)
Propidium iodide, by Thermo Fisher Scientific (1 mentions)
Cd49f pe, by BD (1 mentions)
Cd90 fitc, by BD (1 mentions)
6 protocols using facscalibur c6
1
Phenotypic Characterization of hUC-MSCs
2
Assessing Apoptosis in Cancer Cells
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DU145 and PC3 cells transfected with or without RNF7 shRNA were seeded into 24-well plates in 0.5 ml culture medium (2 × 105 cells/ml). Cells were treated with or without 10 μg/ml cisplatin (P4394, Sigma) for 24 h. Cells were harvested by centrifugation and stained with Annexin V and 1 μl propidium iodide (PI) (V13242, ThemoFisher Scientific) according to the manufacturer’s instruction. Cells were detected by FACSCalibur C6 (BD Biosciences, San Jose, CA, USA) and analyzed using FlowJo software (FlowJo LLC, Ashland, OR, USA).
3
Keratinocyte Phenotyping via Flow Cytometry
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Keratinocytes after 21d cultivation on human feeder layers were trypsinized and 1 × 106 keratinocytes were incubated with each antibody CD49f‐PE and CD90‐FITC for 1 h at room temperature, all the antibodies were purchased from BD (NewJersey, USA). The cells were washed again with PBS and analyzed by flow cytometry (FACS Calibur C6, BD).
4
Cell Cycle Analysis by Flow Cytometry
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Cell cycle analysis was performed using a FACSCalibur C6 flow cytometer (BD Biosciences, San Jose, CA, USA). Cells were fixed in 70% ethanol at 4°C for at least 4 h and then stained with PI/RNase staining buffer (20 µg/ml propidium iodide (PI) containing 10 µg/ml RNase; BD Biosciences, San Jose, CA, USA) for 30 min at room temperature. The DNA distributions in the cells were analyzed by Modifit (v.4.0; Verity Software House, Inc., Topsham, ME, USA) to determine the proportions of cells in each phase of the cell cycle.
5
Annexin V/PI Apoptosis Assay
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Annexin V/ PI staining, cells were treated with or without 1 mM H 2 O 2 (Sigma) for 4 hours. Cells were harvested and stained with Annexin V and 1 µl propidium iodide (PI) (V13242, ThemoFisher Scienti c) according to the manufacturer's instruction. Each sample was detected by FACSCalibur C6 (BD Biosciences) and analyzed using FlowJo software (Treestar).
Computer analysis. Statistical analysis was carried out using the unpaired, two-tailed Student's t-test contained in the Microsoft-Excel 2013 or GraphPad Prism 7. Differences were considered signi cant at a p<0.05.
Computer analysis. Statistical analysis was carried out using the unpaired, two-tailed Student's t-test contained in the Microsoft-Excel 2013 or GraphPad Prism 7. Differences were considered signi cant at a p<0.05.
6
Annexin V/PI Apoptosis Assay
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Annexin V/ PI staining, cells were treated with or without 1 mM H 2 O 2 (Sigma) for 4 hours. Cells were harvested and stained with Annexin V and 1 µl propidium iodide (PI) (V13242, ThemoFisher Scienti c) according to the manufacturer's instruction. Each sample was detected by FACSCalibur C6 (BD Biosciences) and analyzed using FlowJo software (Treestar).
Computer analysis. Statistical analysis was carried out using the unpaired, two-tailed Student's t-test contained in the Microsoft-Excel 2013 or GraphPad Prism 7. Differences were considered signi cant at a p<0.05.
Computer analysis. Statistical analysis was carried out using the unpaired, two-tailed Student's t-test contained in the Microsoft-Excel 2013 or GraphPad Prism 7. Differences were considered signi cant at a p<0.05.
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