Anti cd105 fitc

Cell markers were analyzed following a previously published protocol [11 (link)]. Briefly, cells were washed twice in PBS containing 1% bovine serum albumin (Sigma-Aldrich). The cells were then stained with anti-CD13-FITC, anti-CD14-FITC, anti-CD34-FITC, anti-CD44-PE, anti-CD45-FITC, anti-CD73-FITC, anti-CD90-PE, anti-CD105-FITC, anti-CD106-PE, anti-CD166-PE, or anti-HLA-DR-FITC antibodies (all purchased from BD Biosciences, San Jose, CA). Stained cells were analyzed by a FACSCalibur flow cytometer (BD Biosciences). Isotype controls were used in all analyses.

BM-MSCs and ADSCs were characterized as previously described according to minimal criteria provided by the International Society for Cell and Gene Therapy (ISCT) (Dominici et al., 2006 (link)). The expression of different BM-MSC and ADSC surface markers was determined via flow cytometry as previously described (Cicione et al., 2013 (link)). Cells were trypsinized and resuspended in PBS at 1 × 10⁵ cells/mL incubated for 2 h at room temperature in the dark with the following fluorescein isothiocyanate (FITC) or phycoerythrin (PE) conjugated antibodies: AntiCD105-FITC (#561443, BD Biosciences, Haryana, India), antiCD90-PE (#561969, BD Biosciences), antiCD45-PE (#560976, BD Biosciences), and antiCD73-FITC (#561254, BD Biosciences). A minimum of 25,000 cell events per assay were acquired using CytoFLEX (Beckman Coulter, Brea, CA, United States).

The UC-MSCs were confirmed as MSCs per ISCT recommendations for MSCs. MSCs were observed under an inverted microscope to evaluate their shape. Their phenotypes were evaluated for CD14, CD34, CD44, CD45, CD73, CD90, CD105, and HLA-DR expressions. The protocols for immunophenotyping followed the published protocol [67 ]. Briefly, cells were washed twice in PBS containing 1% bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA). The cells were then stained with anti-CD14-FITC, anti-CD34-FITC, anti-CD44-PE, anti-CD45-FITC, anti-CD73-FITC, anti-CD90-PE, anti-CD105-FITC, or anti-HLA-DR-FITC antibodies (all antibodies were purchased from BD Biosciences, San Jose, CA, USA). Stained cells were analyzed via FACSCalibur flow cytometer (BD Biosciences). Isotype controls were used in all analyses. The purity was calculated based on the percentage of UC-MSCs that were positive for CD44, CD73, CD90, and CD015 and negative for CD14, CD34, CD45, and HLA-DR. The last assay was an in vitro differentiation of the UC-MSCs. This UC-MSC sample was re-evaluated for in vitro differentiation toward adipogenic cells, chondroblasts, and osteoblasts using osteogenic, osteoblast, and chondroblast differentiation kits from Thermo Fisher Company (Thermo-Fisher, Waltham, MA, USA).

Cells were detached with 0.05% trypsin-EDTA and blocked with phosphate buffered saline (PBS) containing 1% fetal bovine serum (FCS; Gibco BRL), 3.3 mg/mL human immunoglobulin (Gammanorm; Octapharma, Stockholm, Sweden) and 0.1% sodium azide. Cells were labelled with different combinations of the following direct conjugated antibodies: anti-CD105-FITC, anti-CD34-FITC, anti-HLA-DR-FITC, anti-CD31-FITC, anti-CD73-PE, anti-CD14-PE, anti-19-PE, anti-HLAclass I-PE, anti-CD146-PE, anti-CD90-APC, anti-CD45-APC (all from BD Bioscience) and anti-CD271-APC (Miltenyi Biotec). Corresponding isotype controls were all from Becton Dickinson. Before analysis, cells were stained with 7-amino-actinomycin D (1 μL/mL; Sigma Aldrich) for dead cell exclusion. Samples were analysed on an FACSCalibur (BD Bioscience). Data acquisition and analysis were performed using CellQuest (BD Bioscience) and FlowJo software (Tree star, Ashland, Oregon, USA).

Cell preparation, antibody staining and flow cytometry were performed as described previously [8 (link)–10 (link), 31 (link)]. Briefly, the MSCs were detached with TrypLE, resuspended in PBS plus 5% BSA (bovine serum albumin, Sigma) and incubated with anti-CD317-PE (Thermo Fisher Scientific), anti-CD73-FITC, anti-CD90-FITC, anti-CD105-FITC, anti-CD45-FITC, anti-CD34-FITC, anti-CD19-FITC, anti-CD11b-FITC, anti-HLADR-FITC, IgG-PE or IgG-FITC (all from BD Biosciences). Data were collected with BD AccuriC6 Plus (BD Biosciences) and analyzed with FlowJo software.

Aliquots of 1 × 10⁵ MSCs were washed and suspended in PBS supplemented with 0.1% FBS. Cells were incubated with anti-CD34-PE, anti-CD45-PE, anti-CD73-PE, anti-CD90-FITC, anti-CD105-FITC, anti-CD80-FITC, anti-CD86-PE, anti-CD154-PE, anti-CD40-PE, anti-HLA-ABC-FITC, or anti-HLA-DR-FITC antibodies (all from BD, San Diego, CA, USA) for 30 minutes at 4°C and then washed twice in PBS containing 0.1% FBS. Cells were then resuspended in 200 μL PBS containing 0.1% FBS and analyzed at 10,000 events per test using the FACSCalibur (BD Biosciences) or NovoCyte (ACEA Biosciences Inc., San Diego, CA, USA). Data were analyzed using NovoExpress 1.2.1 software.

ADSCs of all donors were expanded separately to passage four, pooled, and examined once for surface marker expression using flow cytometry as a pool of six donors. The following monoclonal antibodies conjugated to fluorochromes were used: anti-CD11b-APC, anti-CD13-APC, anti-CD29-PE, anti-CD31-FITC, anti-CD34-FITC, anti-CD44-APC, anti-CD45-FITC, anti-CD63-FITC, anti-CD73-PE, anti-CD90-APC, anti-CD105-FITC, anti-CD106-APC, anti-CD-166-PE, and anti-CD235a (all from Becton Dickinson, Heidelberg, Germany). Isotype antibodies were included for all fluorochromes.
Cells were detached with 0.25% trypsin-EDTA, washed in FACS buffer (1% FCS, 0.1% NaN₃ in PBS), incubated with directly conjugated monoclonal antibodies (5 μl/100,000 cells) in FACS buffer for 30 min on ice, washed twice with FACS buffer, and fixed with 1% paraformaldehyde/PBS. Cells were analyzed using a FACSCanto flow cytometry system (Becton Dickinson). Data acquisition was performed with Diva software (Becton Dickinson) and data were analyzed using FCS express V3 (De Novo Software).