Evaluation of hPDLSCs phenotype was conducted by flow cytometry, as previously described [17 (link),19 (link)]. Briefly, 2.5 × 105 cells were incubated for 30 min with following antibodies: anti-CD44-FITC, anti-CD105-FITC, anti-CD29-PE, and anti-CD45-FITC (Ancell Corporation, Bayport, MN, USA); anti-CD14-FITC (Miltenyi Biotec, Bergisch Gladbach, Germany); OCT3/4-PE, CD73-PE, SOX2-Alexa488, SSEA4-FITC, CD90-FITC (Becton Dickinson, San Jose, CA, USA), and CD34-PE (Beckman Coulter, Fullerton, CA, USA). After incubation with appropriate secondary antibodies, fixation in 1 mL PBS 0.5% paraformaldehyde and washing, cells were analyzed using a FACStar-plus flow cytometry system and the FlowJo™ software v10.0.7 (TreeStar, Ashland, OR, USA).
Facstar plus flow cytometry system
Manufactured by Tree Star
Sourced in United States
The FACStar-plus flow cytometry system is a laboratory instrument designed for analyzing and sorting cells and particles in a fluid suspension. It uses laser technology to detect and measure various physical and fluorescent properties of individual cells or particles as they pass through the system. The core function of the FACStar-plus is to provide researchers with quantitative data on cell populations and their characteristics.
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5 protocols using facstar plus flow cytometry system
1
Phenotypic Characterization of hPDLSCs
2
Phenotypic Characterization of hPDLSCs
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The cytofluorimetric evaluation of the surface markers was carried out on hPDLSCs derived from three different patients and each hPDLSCs was assessed in triplicate. Samples were stained for surface or intracellular antigens (Table 1 ), as previously described by Rajan et al.35 (link). Data were analyzed using a FACStar-plus flow cytometry system and the FlowJo™ software (TreeStar, Ashland, OR, USA).
List of antibodies against surface or intracellular antigens used for cell characterization by cytofluorimetric analysis.
| Antibody | Manufacturer |
|---|---|
| CD44-FITC | Ancell (MN, USA) |
| CD45-FITC | |
| CD29-PE | |
| CD105-FITC | |
| CD14-FITC | Miltenyi Biotec (Bergisch Gladbach, Germany); |
| HLA-DR-PE | Becton Dickinson (San Jose, CA, USA) |
| CD90-FITC | |
| CD73-PE | |
| SOX2-Alexa488 | |
| SSEA4-FITC | |
| OCT3/4-PE | |
| CD34-PE | Beckman Coulter (Fullerton, CA, USA) |
| Fluorescein isothiocyanate (FITC); Phycoerythrin (PE). | |
3
Flow Cytometry Phenotyping of hPDLSCs
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The study of hPDLSC phenotype was performed by flow cytometry, as earlier stated (Diomede et al., 2018a (link)). Shortly, 2.5 × 105 cells were incubated for 30 min with the following antibodies: anti-CD44-FITC, anti-CD105-FITC, and anti-CD29-PE (Ancell Corporation, Bayport, MN, United States); anti-CD14-FITC (Miltenyi Biotec, Bergisch Gladbach, Germany); OCT3/4-PE, SOX2-Alexa488, CD73-PE,CD90-FITC (Becton Dickinson, San Jose, CA, United States) and CD34-PE (Beckman Coulter, Fullerton, CA, United States). After incubation with proper secondary antibodies, fixation in 1 mlL of PBS 0.5% paraformaldehyde and washing, cells were detected utilizing a FACStar -plus flow cytometry system and the FlowJoTM software v10.0.7 (Tree Star, Ashland, OR, United States). The hPDLSCs at P2 were analyzed with an inverted light microscopy Leica DMIL (Leica Microsystem, Milan, Italy).
4
Evaluation of hPDLSCs Differentiation
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Cytofluorimetric detection was performed as previously described by Rajan et al.22 (link) Expression of Oct 3/4, Sox-2, SSEA-4, CD14, CD29, CD34, CD44, CD45, CD73, CD90, and CD105 was evaluated on hPDLSCs. The analysis was performed by using FACStarPLUS flow cytometry system and the FlowJo™ software (TreeStar, Ashland, OR, USA).
To assess the ability to differentiate into osteogenic and adipogenic lineage, hPDLSCs were maintained under osteogenic and adipogenic conditions for 21 and 28 days, respectively, as reported by Cianci et al.23 (link)After the differentiation time, alizarin red and adipo oil red staining were performed on undifferentiated and differentiated cells, in order to evaluate the formation of mineralized precipitates and lipid vacuoles. The observations were carried out at inverted light microscopy Leica DMIL (Leica Microsystem, Milan, Italy). Moreover, the expressions of RUNX-2, ALP, FABP4, and PPARγ genes were evaluated by reverse transcription polymerase chain reaction (RT-PCR) as reported by Cianci et al.23 (link)T-test was used to assess the P value, considering data significant when P < 0.05.
To assess the ability to differentiate into osteogenic and adipogenic lineage, hPDLSCs were maintained under osteogenic and adipogenic conditions for 21 and 28 days, respectively, as reported by Cianci et al.23 (link)After the differentiation time, alizarin red and adipo oil red staining were performed on undifferentiated and differentiated cells, in order to evaluate the formation of mineralized precipitates and lipid vacuoles. The observations were carried out at inverted light microscopy Leica DMIL (Leica Microsystem, Milan, Italy). Moreover, the expressions of RUNX-2, ALP, FABP4, and PPARγ genes were evaluated by reverse transcription polymerase chain reaction (RT-PCR) as reported by Cianci et al.23 (link)T-test was used to assess the P value, considering data significant when P < 0.05.
5
Characterizing hPDLSCs: Cytofluorimetric and Osteogenic Differentiation
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To characterize the hPDLSCs cell population cytofluorimetric detection and osteogenic differentiation procedure were performed as previously reported [24 (link)]. The expression of CD14, CD29, CD34, CD73, CD90 and CD105 was evaluated by means FACStarPLUS flow cytometry system and the FlowJo™ software (TreeStar, Ashland, OR, USA). Then the capacity of hPDLSCs to undergo towards the osteogenic commitment was evaluated maintaining the cell culture under osteogenic conditions with the MSCBM osteogenic kit (Lonza) [25 (link)]. The medium was refreshed every three days. After 3 weeks of differentiation cells were processed and the specific staining was performed using the Alizarin red S solution. The light microscopy Leica DMIL system (Leica Microsystem, Milan, Italy) was used to capture the microphotographs. To validate the differentiation qualitative data, the expressions of osteogenic specific markers (RUNX2 and ALP) were performed by means real time polymerase chain reaction (RT-PCR) and the T-test statistical analyses was used and the data were considered significant when p < 0.05.
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