MSCs were isolated from the iliac crest bone marrow of all the study subjects and purified using density gradient centrifugation as previously described [25 (link)]. After being resuspended in Dulbecco's Modified Eagle's Medium (DMEM, Gibco, USA) containing 10% heat-inactivated fetal bovine serum (FBS, Sijiqing, China), MSCs were seeded in 25 cm2 flasks and cultured in an incubator with 5% CO2 at 37°C. To remove the suspended cells, the medium was replaced 48 hours later and every 3 days thereafter. At 80–90% confluence, the adherent cells were recovered using 0.25% Trypsin containing 0.53 mM EDTA (Gibco, USA) and replated in 75 cm2 flasks as passage 1. MSCs were expanded and used at passages 4 for all experiments. Surface markers of MSCs were detected using flow cytometry (FCM), which was performed on BD Influx cell sorter (BD, USA). CD14-APC, CD29-PE, CD44-FITC, CD45-APC, CD105-FITC, and HLA DR-PerCP antibodies were used (all from BD, USA).
Cd105 fitc
Manufactured by BD
Sourced in United States
CD105-FITC is a cell surface marker that binds to the endoglin protein. It is commonly used in flow cytometry analysis to identify and characterize endothelial cells and their precursors.
Automatically generated - may contain errors
Lab products found in correlation
Cd45 fitc, by BD (25 mentions)
Cd90 fitc, by BD (17 mentions)
Cd73 pe, by BD (15 mentions)
Cd34 fitc, by BD (12 mentions)
Cd73 fitc, by BD (12 mentions)
Cd34 pe, by BD (11 mentions)
Cd29 pe, by BD (10 mentions)
Facscalibur, by BD (9 mentions)
Cd90 pe, by BD (8 mentions)
Cd44 fitc, by BD (7 mentions)
Facscalibur flow cytometer, by BD (7 mentions)
Cd90 apc, by BD (6 mentions)
Cd44 pe, by BD (6 mentions)
Hla dr fitc, by BD (5 mentions)
Cd14 pe, by BD (5 mentions)
Cd146 pe, by BD (5 mentions)
Cd31 pe, by BD (4 mentions)
Cd166 pe, by BD (4 mentions)
Cd45 pe, by BD (4 mentions)
Hla dr percp, by BD (3 mentions)
43 protocols using cd105 fitc
1
Isolation and Characterization of MSCs
2
Characterization of hUCB-MSCs by Flow Cytometry
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hUCB-MSCs were triturated into single cells and labeled with monoclonal mouse anti-human fluorochrome-conjugated antibodies: CD29-PE, CD34-FITC, CD45-FITC, CD105-FITC, CD73-PE, and HLA-DR (BD Bioscience, San Jose, USA). The labeled cells were analyzed via flow cytometry using a FACSCalibur system (BD Biosciences).
3
Autologous Stem Cell Therapy: Infusion Protocol
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At the day of infusion, ASCs monolayer were dissociated as described above and 1 × 106 cells/kg of the donor patient were suspended in 5 ml of saline solution with 50% albumin and 5% ACD (Anticoagulant Citrate Dextrose Solution). Cell suspension was sent to the hospital in cooler with recycled ice.
An aliquot of cells was evaluated after transportation for monitoring viability and phenotype of ASCs by flow cytometry. Cells were washed with PBS-BSA 3% (bovine serum albumin) and incubated at 4°C for 30 min with the following monoclonal antibodies conjugated to fluorescent dyes: CD105-FITC (fluorescein isothiocyanate), CD73-PE (phycoerythrin) and CD90-APC (allophycocyanin), all from BD Biosciences, Franklin Lakes, NJ, USA. Unstained cells were used as controls. Then, cells were washed with PBS-BSA 3% and incubated with 7AAD (7 Amino Actinomycin D). Twenty thousand events were acquired in FACSAria III cytometer (BD Biosciences) and data were analyzed using FACSDiva 8.0 software (BD Biosciences). The percentage of viable cells was estimated by 7AAD exclusion.
Patients that received ASCs were admitted into hospital in the day of the infusion. A single dose of ASCs was infused in a peripheral upper arm vein during 15–20 min. Patients were discharged from hospital 24 h after infusion. Patients started taking oral cholecalciferol 2,000 IU in the same day.
An aliquot of cells was evaluated after transportation for monitoring viability and phenotype of ASCs by flow cytometry. Cells were washed with PBS-BSA 3% (bovine serum albumin) and incubated at 4°C for 30 min with the following monoclonal antibodies conjugated to fluorescent dyes: CD105-FITC (fluorescein isothiocyanate), CD73-PE (phycoerythrin) and CD90-APC (allophycocyanin), all from BD Biosciences, Franklin Lakes, NJ, USA. Unstained cells were used as controls. Then, cells were washed with PBS-BSA 3% and incubated with 7AAD (7 Amino Actinomycin D). Twenty thousand events were acquired in FACSAria III cytometer (BD Biosciences) and data were analyzed using FACSDiva 8.0 software (BD Biosciences). The percentage of viable cells was estimated by 7AAD exclusion.
Patients that received ASCs were admitted into hospital in the day of the infusion. A single dose of ASCs was infused in a peripheral upper arm vein during 15–20 min. Patients were discharged from hospital 24 h after infusion. Patients started taking oral cholecalciferol 2,000 IU in the same day.
4
Mesenchymal Stem Cell Immunophenotyping
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The MSC-like cells were harvested by trypsinization (Gibco) and incubated with 1% bovine serum albumin (BSA) for 1 h at 4 °C to block nonspecific Fc-mediated interactions, then incubated in the dark at 4 °C for 30 min with 400 μl of CD45-FITC (BD Biosciences, 340664, Franklin Lakes, NJ), CD34-APC (BD, 555824), CD105-FITC (BD, 561443), CD73-PE (BD, 561258), and CD90-APC (BD, 559869) antibodies. The cells were stained with PE- or FITC-labeled IgG as an isotype control. The cells were evaluated by a FACSCalibur flow cytometer (Becton–Dickinson, San Jose, CA) and analyzed with FlowJo software (Tree Star Inc., Ashland, Oregon, USA). The percentage of stained cells was calculated relative to the isotype control.
5
Characterization of MSC Surface Markers
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The characteristic cell surface markers for each MSC were evaluated by flow cytometry (Guava EasyCyte Mini Base System, Guava Technologies, Millipore, Hayward, CA, USA). BMSCs and DPSCs were detached with Trypsin-EDTA solution, centrifuged at 1000 rpm for 10 min and fixed with 75% ethanol at −20 °C overnight. The BMSCs and DPSCs were then incubated with the following fluorescent-conjugated antibodies: CD29-FITC, CD34-FITC, CD45-FITC, CD90-FITC, CD105-FITC, and CD146-FITC (BD Biosciences) in PBS for 30 min at 4 °C in the dark. For all of the markers, immunoglobulin-1(IgG1) was used as the isotype control. The cell suspensions were analyzed using a FACSCalibur flow cytometer (Guava EasyCyte Mini Base System, Guava Technologies, Millipore, Hayward, CA, USA). The collected data were further analyzed using FlowJo software (TreeStar Inc., Ashland, OR, USA).
6
Immunophenotyping of Cultured Cells
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After cell detachment using a 0.125% trypsin solution, cells were washed with PBS and resuspended in PBS containing 2% FBS. Cell concentration and viability were monitored using Trypan blue in a Neubauer haemocytometer. The following monoclonal antibodies were used as indicated by the manufacturer (BD Pharmingen): CD90-PE (BD, #555596), CD73-FITC (BD, #561254), CD105-FITC (BD,#561443), CD45-FITC (BD,#347463), CD14-PE (BD,#555398), CD34-PEcy5 (BD,#561819), CD31-PE (BD,#555446), IgG-FITC (BD,#555786), HLA-DR-FITC (BD,#555558), CD166-PE (BD,#560903), CD44-PE (BD,#555479), CD54-PEcy5 (BD,#555512), CD146-PE (BD,# 559263). At least 20,000 events were acquired on a BD FACSCalibur flow cytometer and data was analyzed using CellQuest software.
7
Characterization of BMMSCs Immunophenotype
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BMMSCs were isolated, cultured and induced into osteoblast. The procedure was detailed in our previous studies [32 (link), 33 (link)]. The immunophenotype of BMMSCs were evaluated at P3 (the third passage). After 15 min incubation with the antibodies as follow: CD105-FITC, CD73-FITC, CD29-FITC, CD90-FITC, CD166-PE, CD34-PE, CD45-FITC, CD80-FITC, CD86-FITC (BD Biosciences Pharmingen, San Diego, California, USA), CD44-FITC (Miltenyi Biotec, Bergisch Gladbach, Germany), BMMSCs were analyzed by FACS Calibur system (BD, Mountain View, CA, USA) using Cell Quest software.
8
Characterization of Dental Pulp Stem Cells
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DPSC were donated by the Oral Stem Cell Bank of Beijing, Tason Biotech Co., Ltd. (Beijing, China), derived from three different individuals. Cells were then cultured in α-minimum essential medium (α-MEM, Hyclone, Logan, UT, United States) supplemented with 10% fetal bovine serum (FBS, Gibco, Mulgrave, VIC, Australia), 100 U/mL penicillin, and 100 μg/mL streptomycin (Solarbio, Beijing, China) in a humidified atmosphere of 5% CO2 at 37°C. The cells used in experiments were between passage 3 and 5.
The surface markers of DPSC were identified by flow cytometry. Cells were detached with trypsin/ethylenediaminetetraacetic acid (Gibco) to produce single-cell suspension and resuspended in phosphate buffered saline (PBS) containing 2% FBS. Cells at a concentration of 1 × 106 cells/mL were then incubated with the following monoclonal antibodies: CD34-PE, CD45-PE, CD73-PE, CD90-FITC, CD105-FITC, and CD146-PE (BD Pharmingen, San Diego, CA, United States). The stained cells were analyzed using the flow cytometry system (FC500, Beckman Coulter, Brea, CA, United States) to detect fluorescence intensity and positive rate.
The surface markers of DPSC were identified by flow cytometry. Cells were detached with trypsin/ethylenediaminetetraacetic acid (Gibco) to produce single-cell suspension and resuspended in phosphate buffered saline (PBS) containing 2% FBS. Cells at a concentration of 1 × 106 cells/mL were then incubated with the following monoclonal antibodies: CD34-PE, CD45-PE, CD73-PE, CD90-FITC, CD105-FITC, and CD146-PE (BD Pharmingen, San Diego, CA, United States). The stained cells were analyzed using the flow cytometry system (FC500, Beckman Coulter, Brea, CA, United States) to detect fluorescence intensity and positive rate.
9
Phenotypic Characterization of Adherent AFCs
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Adherent AFCs were separated by trypsin treatment and fixed in 75% ethanol overnight at 4°C. The cells were filtered through a 40 μm mesh and resuspended in fluorescence-activated cell sorting (FACS) buffer [phosphate-buffered saline (PBS) containing 2% fetal bovine serum (FBS) and 0.1% sodium azide]. Directly-conjugated isotype control antibodies, IgG-fluorescein isothiocyanate (FITC) and IgG-phycoerythrin (PE; BD Pharmingen, San Diego, CA, USA), were used as controls to identify the background cells. A total of ~5×105 cells were incubated at 4°C for 40 min with each of the following FITC- or PE-conjugated antibodies (BD Pharmingen): CD133-PE, CD117-PE, CD34-PE, CD105-FITC, CD106-FITC, CD29-PE, CD44-FITC, CD147-FITC and CD90-PE. The cells were subsequently washed in FACS buffer. The antibody-labeled cells were analyzed using a BD FACSCalibur instrument (BD Biosciences, Franklin Lakes, NJ, USA), and data were analyzed using FlowJo version 7.2.5 software (TreeStar, Inc., Ashland, OR, USA). The cell count experiments for the flow cytometry assays were performed in triplicate.
10
Comprehensive Mesenchymal Stem Cell Profiling
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After cell detachment using a 0.125% trypsin solution, cells were washed with PBS and resuspended in PBS containing 2% FBS. Cell concentration and viability were monitored using Trypan blue in a Neubauer hemocytometer. The following monoclonal antibodies were used as indicated by the manufacturers (BD Pharmingen® (BD, Franklin Lakes, New Jersey, USA)): CD90-PE (BD, #555596), CD73-FITC (BD, #561254), CD105-FITC (BD, #561443), CD45-FITC (BD, #347463), CD14-PE (BD, #555398), CD34-PEcy5 (BD, #561819), CD31-PE (BD, #555446), IgG-FITC (BD, #555786), HLA-DR-FITC (BD, #555558), CD166-PE (BD, #560903), CD44-PE (BD, #555479), CD54-PEcy5 (BD, #555512), CD146-PE (BD, #559263). Isotype controls were used for determining nonspecific binding and defining cut-off values. A minimum of 20,000 events were acquired on a BD FACS Calibur® flow cytometer and results were analyzed using CellQuest™ software.
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