At P3, cells were harvested by trypsinization with 0.05% trypsin (Invitrogen) upon reaching 90% confluence, and re-suspended in DPBS to reach a final cell density of 1.5 × 106 cells/ml. An amount of 200 μl of cell suspension (1 × 105 cells) was incubated in the dark for 1 hr at 37°C with Phycoerythrin-conjugated antibodies against CD44, CD73, CD166, CD105 and CD34, and fluoro isothycyanate-conjugated antibodies against CD45 and HLA-DR (all from BD Pharmingen) for specific surface antigens analysis by using flow cytometer. Excess antibodies were removed by washing with DPBS. All analyses were standardized against negative control cells incubated with Isotype-specific IgG1-PE and IgG1-FITC (BD Pharmingen). At least 10,000 events were acquired on Guava Technologies flow cytometer, and the results were analysed by using Cytosoft, Version 5.2, Guava Technologies.
Cytosoft version 5
Manufactured by Merck Group
Sourced in United States
Cytosoft, Version 5.2, is a software application designed for cell analysis. It provides tools for the visualization, measurement, and analysis of cellular data.
Automatically generated - may contain errors
Lab products found in correlation
Cd73 pe, by BD (3 mentions)
Hla dr fitc, by BD (3 mentions)
Trypsin, by Thermo Fisher Scientific (2 mentions)
Isotype specific igg1 pe and igg1 fitc, by BD (2 mentions)
Igg1 pe, by BD (2 mentions)
Igg1 fitc, by BD (2 mentions)
Cd90 phycoerythrin pe, by BD (2 mentions)
Cd45 fluoroisothyocyanate fitc, by BD (2 mentions)
Cd166 pe and cd34 pe, by BD (2 mentions)
Guava technologies, by Merck Group (2 mentions)
Phycoerythrin conjugated antibodies against cd44, by BD (1 mentions)
Cd34 pe, by BD (1 mentions)
Cd44 pe, by BD (1 mentions)
Cd166 pe, by BD (1 mentions)
35mm tissue culture dish, by BD (1 mentions)
Propidium iodide, by Roche (1 mentions)
Fitc conjugated antibody, by BD (1 mentions)
Facscalibur cytometer, by BD (1 mentions)
Dulbecco s pbs dpbs, by Thermo Fisher Scientific (1 mentions)
Propidium iodide rnase staining buffer, by BD (1 mentions)
8 protocols using cytosoft version 5
1
Mesenchymal Stem Cell Surface Marker Profiling
2
Immunophenotyping of Cell Populations
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Fluorescence activated cell sorting (FACS) was carried out as described previously in our paper [20 (link)]. The following antibodies were used to mark the cell surface epitopes: CD90-phycoerythrin (PE), CD44-PE, CD73-PE, CD166-PE and CD34-PE, CD45-fluoroisothyocyanate (FITC), and HLA-DR-FITC (all from BD Pharmingen). All analyses were standardized against negative control cells incubated with isotype specific IgG1-PE and IgG1-FITC (BD Pharmingen). At least 10,000 events were acquired on Guava Technologies flow cytometer, and the results were analyzed using Cytosoft, Version 5.2 (Guava Technologies).
3
Immunophenotyping of Pb2+ Exposed Cells
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Pb2+ exposed cell lines were subjected to immunophenotyping analysis by using a flow cytometry. A total volume of 200 μL of a cell suspension (1 × 105 cells) was incubated with the labeled antibodies in the dark for 1 hour at 37°C. The following antibodies were used to mark the cell surface: epitopes-CD90-phycoerythrin (PE), CD44-PE, CD73-PE, CD166-PE and CD34-PE, and CD45-Fluoro-isothyocyanate (FITC), and HLA-DR-FITC (all from BD Pharmingen). All the analyses were standardized against a negative control of cells incubated with isotype-specific IgG1-PE and IgG1-FITC (BD Pharmingen). At least 10,000 events were acquired on Guava Technologies flow cytometer and the results were analyzed using Cytosoft, Version 5.2, from Guava Technologies.
4
Cell Cycle Analysis by Flow Cytometry
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Cells were seeded at 5000 cells/cm2 on a 35-mm tissue culture dish (BD Pharmingen) and cultured until 90% confluency. The cells were then detached, fixed, and permeabilized in 70% ethanol overnight in the dark at 4 °C. After that, the cells were treated with RNase A (1 mg/ml final concentration), and stained with propidium iodide (PI, Roche, 50 μg/ml final concentration). DNA content was analyzed on Guava Technologies (Millipore, Billerica, MA) flow cytometer using Cyto- soft, Version 5.2, Guava Technologies software.
5
Characterization of Mesenchymal Stem Cells
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MSCs at passage 3 were digested and resuspended in 100 μL of PBS with a density of 1 × 105 cells. The cells were labeled with FITC-conjugated antibodies (BD Biosciences, San Diego, USA), including CD13, CD34, CD45, CD73, CD90, and CD105. FITC-conjugated isotype-matching IgS was used to determine nonspecific staining. The cells were examined using a FACSCalibur cytometer (BD Biosciences), and the data were analyzed using Cytosoft version 5.2 (Guava Technologies, Hayward, CA, USA).
6
Cell Cycle Analysis by Flow Cytometry
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The cells were pre-seeded on a 35-mm tissue culture dish (BD Pharmigen) at a density of 5000 cells/cm2. Upon reaching 90% confluence, the cells were detached, fixed and permeabilized in 70% ethanol and left overnight at 4°C. Thereafter, 500 μl was extracted (containing 1 × 106 cells), and DNA was stained with Propidium iodide/RNAse staining buffer (BD Pharmigen) for 15 min. at room temperature and subsequently washed in Dulbecco's PBS (DPBS; Invitrogen). DNA content was analysed on Guava Technologies (Millipore, Billerica, MA, USA) flow cytometer by using Cytosoft, Version 5.2, Guava Technologies software.
7
Immunophenotyping of Cultured Cells
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The immunophenotyping characterization of the cells were done using flow cytometry at sub-culture 5. After reaching 90% confluency, the cells were trypsinized with 0.05% trypsin (Invitrogen) and re-suspended in DPBS (Invitrogen) at a cell density of 1.5×106 cells/mL. A total of 200 µL of the cell suspension (1 x 105 cells) was incubated with labeled antibodies in the dark for one hour at 37°C. The following antibodies were used: CD90-phycoerythrin (PE), CD44-PE, CD73-PE, CD166-PE and CD34-PE, CD45-Fluorescein-isothyocyanate (FITC) and HLA-DR-FITC. FITC- or PE-labeled isotype-matched immunoglobulins were used as negative controls. The stained cells were analyzed using Guava Technologies flow cytometer and the results were analyzed using Cytosoft, Version 5.2, Guava Technologies. All antibodies were purchased from BD Pharmingen.
8
Immunophenotyping of Deciduous DPSCs
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Immunophenotyping of deciduous DPSCs were examined using flow cytometry at passage 5 [22 (link)]. The antibodies used to mark the cell surface epitopes were CD90-phycoerythrin (PE), CD73-PE, CD166-PE and CD34-PE, CD45-fluoroisothiocyanate (FITC), and HLA-DR-FITC (all from BD Pharmingen, San Jose, CA, USA). All analyses were standardized against negative control cells incubated with isotype-specific immunoglobulin (Ig) G1-PE and IgG1-FITC (BD Pharmingen). At least 10,000 events were acquired on a Guava Technologies flow cytometer, and the results were analysed using Cytosoft, Version 5.2 (Guava Technologies, Hayward, CA, USA).
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