Cd90 apc

Each assay contained a unique combination of the following mouse anti-human monoclonal antibodies: CD73-FITC, CD90-APC, CD105-PE (BioLegend, San Diego, CA, USA), CD34-PE, CD36-APC, CD146-PE (Miltenyi BioTech, Bergisch, Germany), CD61-PE (Beckman Coulter Inc., Pasadena, CA, USA), CD15-FITC, and 7-AAD (Becton Dickinson, Franklin Lakes, NJ, USA). All antibodies were titrated and used at a 50 ng/assay concentration. Isotype controls and specific mAbs were employed at the same final concentrations. Routinely, 50,000 hASCs (in 100 µL FACS buffer) were mixed with the appropriated antibody combination and incubated for 15 min at RT in the dark. Finally, the sample was diluted with 100 µL FACS buffer before the acquisition, according to our flow cytometer procedure (see Section 2.3.3).

Surface markers on MSC-EVs were analyzed using Apogee A50-Micro flow cytometer (Apogee Flow Systems, UK). The following fluorochrome-conjugated antibodies against murine antigens were used according to the manufacturer’s protocols: CD29-APC (clone: HMβ1-1, Biolegend), CD44-APC (clone: IM7, Biolegend), CD81-APC (clone: Eat2, BD Bioscence), CD90-APC (clone: 30-H12, Biolegend), CD309-APC (clone: Avas12, Biolegend) and Sca-1-APC (clone: E13-161.7, Biolegend) as well as the following isotype controls: Armenian hamster IgG-APC (clone: HTK888, Biolegend), Rat IgG2a, κ-APC (clone: RTK2758, Biolegend) and rat IgG2b, κ-APC (clone: RTK4530, Biolegend). MSC-EVs were co-stained with SYTO RNA Select dye (ThermoFisher Scientific), which binds RNA molecules. Staining was conducted for 30 min in the dark at 4 °C. The obtained results were analyzed using Apogee Histogram software (Apogee Flow Systems). In order to confirm the presence of the indicated antigens on MSC-EVs, an ImageStream X Mark II imaging cytometer (Merck) was additionally used.

To assess the immunophenotype of the cells, we first washed the cells with PBS and detached them using 0.25% trypsin‐EDTA. All of the cells were then fixed with 4% paraformaldehyde at 4°C for 18 h, after which the paraformaldehyde was discarded after centrifugation at 400 × g for 3 min. The fixed samples were then washed three times with a solution of 2% FBS in PBS. Flow cytometry analyses were conducted with the following primary antibodies: OCT4‐FITC (Biolegend), SOX2‐FITC (Biolegend), CD90‐APC (Biolegend), CD73‐PE (Biolegend), CD105‐PE (Biolegend), CD11b‐PE (Biolegend), CD34‐PerCP (Biolegend), CD45‐PE (Biolegend), and HLA‐DR‐APC (Biolegend). These antibodies were used at a 1:100 dilution and incubated at 4°C for 1 h in a dark environment. Afterward, the cells were washed three times with a 2% FBS in PBS solution, and fluorescence signals were detected using a BD Accuri C6 flow cytometer (BD Science, USA).

Human adipose tissue-derived stem cells were provided from the stem cell technology research center, Bonyakhteh institute, Tehran, Iran. The cells were cultured in a T-75 flask and were maintained in DMEM/F12 (Gibco) supplemented with 10% FBS (Gibco) and 100 U/ml penicillin and 100 µg/ml streptomycin (Invitrogen) at 37 °C and 5% CO₂. The medium was replaced every three days. Characterization of the stem cells was performed by optical microscopy and flow cytometry.
For immunophenotypic characterization second passage of ADSCs were used. ADSCs were stained with conjugated antibodies including CD105-PE (eBioscience; USA), CD90-APC (BioLegend; USA), CD73-PE-Cy7 (BioLegend; USA), and CD45-FITC (BioLegend; USA). The cells were then trypsinized and the suspension was centrifuged at 300 g for 4 min. A total of 5 × 10⁵ cells were dissolved in 0.2 ml PBS and incubated with conjugated antibodies for 20 min in a dark room^{27 (link)}. The samples were analyzed using a flow cytometer (BD FACSVerse, BD Biosciences, USA) for identification of specific fluorescence channels of each antibody.

Cell surface antigens on cells were evaluated with FACS. The cells were dissociated with 0.05% trypsin/EDTA (Highclone), washed with PBS, fixed with 4% paraformaldehyde, permeabilized with Triton X‐100, and blocked with a BSA mixture. The samples were then stained with antibodies against human octamer‐binding transcription factor 4 (OCT4; R&D systems, 1:200), stage specific embryonic antigen 1 (SSEA1; R&D systems, 1:200), cluster of differentiation 31 (CD31‐FITC; Miltenyi Biotec, 1:100), CD34 (CD34‐PerCP, BioLegend, 1:100), human leukocyte antigen ‐ DR isotype (HLA‐DR, HLA‐DR‐APC, BioLegend, 1:100), CD73 (CD73‐PE, BioLegend, 1:100), CD90 (CD90‐APC, BioLegend, 1:200), CD105 (CD105‐FITC, BioLegend, 1:150), integrin α5 (Santa Cruz, 1:200), integrin α11 (Abcam, 1:200), integrin β1(Abcam, 1:200), and integrin β5 (BioLegend, 1:200) for 30 min or 1 h at 4 °C. Samples were subsequently stained with fluorescently labeled secondary antibodies (Alexa Fluor‐488 or 594 conjugated goat antirabbit (Abcam, 1:400), Alexa Fluor‐488 or 594 conjugated goat antimouse (Abcam, 1:400)) for 30 min at 4 °C. The corresponding mouse/rabbit isotype antibodies (Abcam, 1:200) were used as controls. Cell immunotypes were determined with the Accuri C6 flow cytometer (BD Biosciences) and the percentage of expressed cell surface antigens was calculated for 10 000 gated‐cell events.

DPSCs were treated with 0.25% trypsin/0.02% EDTA (Sigma-Aldrich) and centrifuged to generate a cell pellet. Cells were labelled with 10 μL FcR blocking solution (Miltenyi Biotec, Bergisch Gladbach, Germany) and various antibodies (see below, 10 μL per 1 × 10⁶ cells unless stated) in a total volume of 100 μL labelling buffer (PBS, 2 mM EDTA (Alfa Aesar, Heysham, UK) and 0.5% (w/v) BSA (Sigma-Aldrich)) for 20 minutes at room temperature in the dark. Following labelling, 900 μL labelling buffer was added to each sample before centrifugation and resuspension in 500 μL labelling buffer. Samples were then analysed using an LSRII flow cytometer (BD Biosciences, San Jose, CA) running FACSDiva 8.0 software (BD Biosciences), subsequent data analysis was performed using Kaluza 1.3 (Beckman Coulter, Inc., Miami, FL). Antibodies used were: CD29-Alexa Fluor 488 (5 μL per 1 × 10⁶ cells), CD34-FITC (5 μL per 1 × 10⁶ cells), CD44-FITC, CD73-PE (2 μL per 1 × 10⁶ cells), CD90-APC and CD166-PE (all Biolegend, San Diego, CA). All threshold values were obtained by analysing autofluorescence of non-labelled hDPSCs and reactivity of isotype matched controls. Events were gated based on forward and side scatter and colour compensation was performed.