SHED and MSC-like cells from iPS-SHED and from iPS-FIB were harvested and resuspended to 105 cells in 100 uL of PBS containing 1% BSA. Cells were separately labeled with FITC, PE, PE-Cy5, PERCP-Cy5.5, or APC-H7 conjugated rat anti-human antibodies CD29, CD31 (Biolegend), CD34, CD45, CD73, CD90 CD105, and CD166 (Becton Dickinson) on ice and protected from light for 40 min. An isotype-matched mAb was used as a control (Becton Dickinson). Data were acquired and analyzed with the FACSAria II cytometer and CellQuest software (Becton Dickinson). Multipotential differentiations of MSC-like cells from iPS-SHED and from iPS-FIB were performed as previously described by de Mendonça Costa et al., 2008 [13 (link)], and representative pictures of adipogenesis, osteogenesis, and chondrogenesis were included as supplementary Figure 2.
Cd166
Manufactured by BD
Sourced in United States
CD166 is a cell surface marker that is commonly used in flow cytometry and cell sorting applications. It is a member of the immunoglobulin superfamily and is involved in cell-cell adhesion. CD166 is expressed on a variety of cell types, including hematopoietic stem cells, neural stem cells, and certain cancer cell types.
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Lab products found in correlation
Cd105, by BD (17 mentions)
Hla dr, by BD (11 mentions)
Cd11b, by BD (4 mentions)
Cd146, by BD (3 mentions)
Cd106, by BD (3 mentions)
Facsverse, by BD (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Cellquest software, by BD (2 mentions)
Cd146, by Miltenyi Biotec (2 mentions)
Ghost red viability dye, by Cytek Biosciences (2 mentions)
Kaluza analysis software, by Beckman Coulter (2 mentions)
Cd105, by BioLegend (2 mentions)
Cytoflex s, by Beckman Coulter (2 mentions)
Lsr 2 flow cytometer, by BD (2 mentions)
Cd133, by BD (2 mentions)
Cd49a, by BD (2 mentions)
Cd49e, by BD (2 mentions)
Cd151, by BD (2 mentions)
Hla dr dp dq, by BD (2 mentions)
Facsaria 3 cell cytometer, by BD (2 mentions)
36 protocols using cd166
1
Characterization of Stem Cells from iPS
2
Characterization and Isolation of Umbilical Cord-Derived Stem Cells
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Cells from the digested cord tissue and cultured in MSCGM were characterized as UCT-MSC based upon morphology and phenotype determined by flow cytometry as separate biological replicates. Antibodies used for phenotypic characterization included CD31, CD14, CD90, CD44, CD83, CD105, CD45, UEA-1, Stro-1, ICAM-1, CD146, CD11b, CD29, CD80, CD117, CD166, CD34, and CD309 (Becton Dickinson, Franklin Lakes, NJ, United States). UEA-1 was purchased from Sigma-Aldrich (St. Louis, MO, United States) and CD51 was purchased from Immunotec (Beckman-Coulter, Miami, FL, United States).
To obtain cord tissue-derived endothelial progenitor cells (UCT-EPC), cells from the digested cord tissue that had been cultured in EGM-2 were labeled with FITC-conjugated Ulex europaeus agglutinin 1 (UEA-1) (Sigma-Aldrich, St. Louis, MO, United States) and isolated with anti-FITC microbeads (MACS Miltenyi Biotec, San Diego, CA, United States). Cells were then phenotypically defined by flow cytometry as separate biological replicates. Endothelial cell lineage was confirmed by visualizing tube formation with the Cultrex “In Vitro Angiogenesis Assay Tube Formation Kit” (Trevigen, Gaithersburg, MD, United States). UCT-EPC were cultured on gelatin-coated flasks using EGM-2 growth media in a humidified 37°C incubator at 5% CO2.
To obtain cord tissue-derived endothelial progenitor cells (UCT-EPC), cells from the digested cord tissue that had been cultured in EGM-2 were labeled with FITC-conjugated Ulex europaeus agglutinin 1 (UEA-1) (Sigma-Aldrich, St. Louis, MO, United States) and isolated with anti-FITC microbeads (MACS Miltenyi Biotec, San Diego, CA, United States). Cells were then phenotypically defined by flow cytometry as separate biological replicates. Endothelial cell lineage was confirmed by visualizing tube formation with the Cultrex “In Vitro Angiogenesis Assay Tube Formation Kit” (Trevigen, Gaithersburg, MD, United States). UCT-EPC were cultured on gelatin-coated flasks using EGM-2 growth media in a humidified 37°C incubator at 5% CO2.
3
Stemness Confirmation of Wharton's Jelly Mesenchymal Stem Cells
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In order to confirm the stemness of WJ-MSCs, cell surface marker analysis was performed. Harvested MSCs were washed in PBS supplemented with 2% FBS in order to block for non-specific binding sites. Immunophenotypic analysis of MSCs was carried out using flow cytometry for the following markers: CD73, CD90, CD105, CD166, CD14, CD11b, HLA-DR (MHCII), CD34, CD45 and CD19 (BD Biosciences, USA). At least 10,000 events were acquired by using the BD FACS Verse flow cytometer, and the results were analyzed by using the BD FACSuite software version 10. Flow cytometry for appropriate isotype controls were also performed.
4
Immunophenotyping of Primed and Naïve MSCs
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Flow cytometric analysis was performed on P3 naïve and primed IFP (n = 3) and BM (n = 3) MSC. 2.0 × 105 cells were labelled with monoclonal antibodies specific for: CD10, CD44, CD56, CD73, CD90, CD105 (Biolegend, San Diego, CA), CD146, LepR (Miltenyi Biotec, Auburn, CA), CD166, CD271, NG2 (BD Biosciences, San Jose, CA), CD200, CXCR4 (Invitrogen) and the corresponding isotype controls (Supplementary Table S2 ). All samples included a Ghost Red Viability Dye (Tonbo Biosciences, San Diego, CA). Data were acquired using a Cytoflex S (Beckman Coulter, Brea, CA) and analysed using Kaluza analysis software (Beckman Coulter).
5
Multiparametric Phenotypic Analysis of Cells
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After a 5 min dissociation step with TrypLE™Select at 37 °C, cells were washed with DPBS by centrifugation and a total of 2–3 × 105 cells were used per analysis. For membrane markers, cells were incubated with the primary antibody for 1 h and with the secondary antibody for 30 min at 4 °C in DPBS with 5% (v/v) FBS. For intracellular markers, cells were permeabilized using the Inside Stain Kit (Miltenyi Biotec) according to the manufacturer‘s instructions. The following primary antibodies were used: SirPα/β (1:20, CD172a/b-PE, BioLegend), Troponin T (1:200, TnT, Thermo Scientific), CD105 (1:20,BD Pharmingen), CD166 (1:20,BD Pharmingen), CD44 (1:5, eBiosciences), CD11b (1:10, AbDSerotec), CD34 (1:5, BD Pharmingen), CD45 (1:5, BD Pharmingen), and isotype controls mouse IgG1k (1:5, BD Pharmingen), mouse IgG1 (1:2.5,Santa Cruz Biotechnologies), and rat IgG2b (1:5, eBiosciences). Cells were analyzed in a CyFlow® space (Partec GmbH) instrument, registering at least 10,000 events/sample.
6
Flow Cytometry Analysis of MSC
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FACS analysis was performed as described earlier [32 (link)]. Briefly, 105 MSC in 100 µl PBS were incubated with fluorochrome-labelled antibodies against CD90, CD29 (eBiosciences, San Diego, CA, USA), CD105 (BioLegend, San Diego, CA, USA), CD73, CD166, CD45 (BD Pharmingen, Heidelberg, Germany), C3aR (AbD Serotec, Puchheim, Germany) or the according isotype controls for 30 minutes in the dark on ice. After washing MSC twice with PBS, MSC were resuspended in 300 µl PBS and analyzed immediately on the FACSCalibur flow cytometer (Becton Dickinson, Heidelberg, Germany) with dual-laser technology and the CellQuest software V. 5 (Becton Dickinson). A minimum of 1.5*104 cells were acquired.
7
Characterization of SHED by Flow Cytometry
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SHEDs were obtained from the Sichuan Neo-life Stem Cell Biotech Inc, China. They were from the exfoliated deciduous of 6 -8 years old children and cultured in Dulbecco's modified Eagle medium (DMEM; Gibco, Grand Island, NY, USA) containing 10% fetal bovine se-rum (Gibco) and antibiotics (100 U/mL penicillin, 100 μg/mL streptomycin; Gibco) at 37°C and in 5% CO 2 atmosphere.
Flow cytometry was performed to characterize SHED. 10 6 cells from cultivated human exfoliated teeth in the 5 th passage were incubated with the following conjugated antibodies against human cell surface molecules: CD90, CD105, CD166, CD73,CD29, CD44, CD34, CD79α, and HLA-DR (PharMingen-BD Biosciences, USA), conjugated with FITC (Santacruz, USA) or PE (PharMingen-BDBiosciences, USA). Data acquisition was performed using the FACSAria III flow cytometer (BD Biosciences, USA) and 10,000 events were analyzed using FACS Diva 6.1.3 software (BD Biosciences, USA).
Flow cytometry was performed to characterize SHED. 10 6 cells from cultivated human exfoliated teeth in the 5 th passage were incubated with the following conjugated antibodies against human cell surface molecules: CD90, CD105, CD166, CD73,CD29, CD44, CD34, CD79α, and HLA-DR (PharMingen-BD Biosciences, USA), conjugated with FITC (Santacruz, USA) or PE (PharMingen-BDBiosciences, USA). Data acquisition was performed using the FACSAria III flow cytometer (BD Biosciences, USA) and 10,000 events were analyzed using FACS Diva 6.1.3 software (BD Biosciences, USA).
8
Phenotypic and Functional Characterization of WJ-MSCs
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The WJ-MSCs (passage 2) were detached using 0.25% EDTA-Trypsin solution, and were harvested in a 15-mL conical tube. After centrifugation, the WJ-MSCs were washed and resuspended in phosphate-buffered saline with 2% FBS to block the nonspecific binding sites. According to the MSC criteria of the International Society for Cell Therapy [2 (link)], an immunophenotypic analysis of the WJ-MSCs was performed via flow cytometry analysis, using the following markers: CD11b, CD14, CD19, CD44, CD45, CD73, CD90, CD105, CD166, and HLA-DR (BD Biosciences, Franklin Lakes, NJ, USA). At least 10.000 events were acquired on a BD FACSVerse (BD Biosciences), and the results were analyzed with BD FACSuite software v.10 (BD Biosciences).
The differentiation of the WJ-MSCs was tested according to the procedure outlined in a previous report [49 (link)].
The differentiation of the WJ-MSCs was tested according to the procedure outlined in a previous report [49 (link)].
9
Immunophenotypic Characterization of PDLSCs and GMSCs
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The 5th passage (P5) of PDLSCs and GMSCs were used for this experiment. After filtering through a cell strainer 70 μm sieve, washed with PBS and blocking buffer (2% FBS in PBS), counted 1 × 106 cells, and then added 5 μL CD34 (BD Biosciences, Catalog No: 555822), CD49d (BD Biosciences, Catalog No: 555503), CD90 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), CD106 (BD Biosciences, Catalog No: 565417) and CD166 (BD Biosciences, Catalog No: 565461), incubated at room temperature for 30 min in the dark, and washed the cells twice with PBS after 30 min to remove non-specific antibody binding. In addition, the cell suspension only added with PBS was used as the experimental control group. Samples were analyzed with flow cytometer BD FACSCanto™ Clinical software. The data results were analyzed and drawn with BD FACSDiva™ Software (BD Biosciences).
10
Immunophenotypic Characterization of MISB10 Cells
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MISB10 cells cultured in T75 flasks were treated with Accutase (BD Biosciences) and washed twice with PBS with 1% BSA. Suspensions were counted and measured for cell viability using the Vi-Cell XR cell counting and viability analyzer (Beckman Coulter). Cells were diluted in PBS to 1 × 106 cells/ml and stained for 30 min at RT with membrane viability dye (LIVE/DEAD Fixable Near-IR, Invitrogen). Cells were washed and distributed to a 96-well plate containing staining antibodies and Hoechst 33342 (Invitrogen). For characterization panels, immunophenotyping antibodies to the following targets were used: HER-2/neu, CD24, CD29, CD44, CD45, CD49f, CD90, CD166, CD326 (BD Biosciences), and CD133 (Miltenyi Biotec, Auburn, CA). Cells were incubated for 30 min at RT in the dark and then rinsed twice in PBS before acquisition with a BD LSRII flow cytometer. Analysis of results was performed using FACSDiva v6.1.3 and FlowJo analysis software (FlowJo).
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