Cd105 fitc

AT-MSC were isolated from subcutaneous adipose tissue of five healthy donors (2 females/3 males). The age of the donors was between 34-58 years old. The tissue was mechanically disrupted and enzymatically digested with 0.5 mg/ml collagenase type IV (Sigma-Aldrich, St. Louis, MO) in RPMI for 30 min at 37°C under continuous shaking. Thereafter, the cells were resuspended in MEM-α with 10% fetal bovine serum (FBS; Lonza, Verviers, Belgium), 2 mM L-glutamine and 1% P/S, filtered through a 100 µm cell strainer, and transferred to 175 cm² culture flasks (Greiner Bio-one, Essen, Germany). At 90% confluence AT-MSC (passage 2-6) were collected to generate MP. The phenotypic characterization of AT-MSC was performed by flow cytometry using FACSCANTO-II with FACSDIVA Software (BD Biosciences, San Jose, CA). AT-MSC were incubated with mouse-anti-human monoclonal antibodies against CD13-PE-Cy7; HLA-DR-PERCP; HLA-ABC-APC; CD31-FITC; CD73-PE; PD-L1-PE (all BD Biosciences); CD90-APC and CD105-FITC (R&D Systems, Abingdon, UK). All the antibodies were incubated with the cells for 30 min, at room temperature in the absence of light.

To confirm the phenotype of the undifferentiated DPPSC, FACS analysis was performed. The following fluorochrome labelled monoclonal antibodies were used: CD105-FITC (R&D Systems), CD29-PE (R&D Systems), CD146-FITC (BD Pharmingen), CD45-PE (BD Pharmingen), NANOG-FITC and OCT3/4-FITC (R&D Systems). To analyze the control samples, different IgG isotypes coupled to PE and FITC fluorochromes (BD Pharmingen) were used. The cells were suspended in PBS with 2% FBS and were incubated for 45 min at 4 °C in the absence of light. Subsequently, the cells were washed twice with 2% FBS-PBS and centrifuged for 6 min at 1800 rpm, thereby removing any residual fluorochrome to avoid false positive results. The pellets were re-suspended in volumes between 300 and 600 μl (depending on the number of cells) of PBS with 2% FBS. The flow cytometry measurements were made using a FACS cytometer (FACS Calibur, BD Biosciences) and analyzed with WinMDI 2.8 software. To detect and exclude nonspecific unions and auto fluorescence, at least 5x10⁵ cells were used for each sample.

MSC phenotype was evaluated using P3 and P10 MSCs by the expression of CD34-APC (BD Biosciences), CD90–PE (BD Biosciences), CD73-PE (BD Biosciences) and CD105-FITC (R&D Systems, Minneapolis, MN). As previously described^{30 (link)}, cells were incubated for 30 min at room temperature with antibodies, and flow cytometry was performed on an LSR-II flow cytometer (Beckton Dickson). Isotype matched controls were used to set the electronic gates on the flow cytometer. The data were analyzed using FlowJo software (Tree Star).

For each marker, 10,000 MSCs were stained as described previously [20 (link)]. Cells were detached using TrypLE Express (Gibco) recombinant trypsin. TrypLE was deactivated using phosphate-buffered saline+2% FCS. Cells were incubated for 30 min at 4°C in the dark with human FcR blocking reagent (Miltenyi Biotec, Leiden, NLD) and the following antibodies: CD45-PE (#560975 BD Pharmigen, Breda, NLD), CD14 (#R0864, Dako, Heverlee, BEL), CD19 (130-091-328, Miltenyi), CD34 (BD #555821), CD73 (BD #550257), CD90 (#B113673 Biolegend, Fell, DE), CD105-Fitc (FAB 10971F, R&D, Minneapolis, MN, USA), and CD140b (BD #558821). Subsequently, cells were washed with PBS+2% FCS. Cell fluorescence was measured on a FACSCanto II flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). SYTOX Blue (Molecular Probes/Invitrogen, Eugene, OR, USA) was used for exclusion of dead cells.

The immunophenotypic features of the isolated cells were confirmed by flow cytometry. Briefly, the cells were dissociated with trypsin/EDTA; then, cell suspensions were stained using various antibodies against MSC markers including CD105-FITC (R&D Systems), CD90-PE (Dako), CD73-PE (Abcam), CD45-FITC (Dako), and CD34-PE (Biosciences) and secondary goat antimouse IgG-PE. The given cells were also labeled with isotype-matched antibodies and subsequently served as background controls. Finally, the cells were treated with suitable secondary antibodies, and the samples were submitted to the FACSCalibur cytometer (Becton Dickinson). Data analysis was performed using the CELL QUEST software.

hUCMSCs were starved in serum-free DMEM-LG medium containing 0.1% BSA for 16 hours and stimulated with or without 100 ng/ml IL-1β for 36 hours. Cells were washed twice with PBS and detached by HyQtase (Thermo, Logan, UT). Cell were then suspended 1*10⁶ cells in 100 μl PBS and incubated with 1:20 dilution of conjugated antibodies CD105-FITC (R&D systems, USA), CD34-FITC (Becton, Dickinson and Company, USA), CD73-PE (Becton, Dickinson and Company, USA), CD45-PE (Thermo Fisher Scientific, USA), CD90-PE (Beckman Coulter, USA) at 4°C for 30minutes on the shaker. The unstained cells were cultured with PBS only. Cells were then washed twice with PBS and centrifuged at 0.3 g for 5 minutes. Finally, cells were re-suspended in 200 μl PBS and immediately analyzed using Beckman Coulter CytoFLEX (Beckman, IL, USA).

MSC phenotype was evaluated after 2 weeks’ expansion by the expression of CD34-APC (BD Biosciences Pharmingen, San Diego, CA), CD45-FITC (BD Biosciences Pharmingen, San Diego, CA), CD90–PE (Dako Cytomation Denmark A/S, Glostrup, Denmark), CD73-PE (BD Biosciences Pharmingen) and CD105-FITC (RD Systems, Minneapolis, MN). Cells were incubated for 30 min at room temperature with antibodies and flow cytometry was performed on a LSR-II flow cytometer (Beckton Dickson). Isotype matched controls were included for each antibody and used to set the electronic gates on the flow cytometer. The data were analyzed using FlowJo software (Tree Star).