Hla dr

For the detection of the immunophenotype of the CEM-C7/HDR and CEM-C7–14 cells, we used antibodies against the following targets: CD33, CD34, HLA-DR, cyTdT, cCD3, CD3, CD4, CD5, CD7, CD8, CD117, CD1α, CD2, CD10, CD19, CD20, CD13 and CD45 (Becton Dickinson Inc., Franklin Lakes, NJ, USA). Positivity for the antigens was determined using a FACSCalibur flow cytometer (Becton Dickinson Inc.).

Cell surface marker analysis was performed by flow cytometry (Miltenyi Biotec) to confirm the stem cell characteristics of ADSCs. The cell surface markers used were CD73 allophycocyanin (APC), CD90 fluorescein isothiocyanate (FITC), and CD105 peridinin-chlorophyll-protein (PerCP) Cy5.5 as positive MSCs markers, and lineage negative marker-PE including CD34, CD45, CD11b, CD19, and human leukocyte antigen (HLA)-DR (Becton Dickinson) as positive haematopoietic cells markers. The cells (1 × 10⁵, passage 3) were stained with fluorescence-labelled probes specific to cell surface molecule. Data were obtained from 10,000 events per analysis.

The antibodies for surface marker analysis for MSCs or isolation of CD146+MSCs are PEcy7-conjugated anti-Sca-1, CD105, CD90, CD45, CD34, HLA-DR and FITC-conjugated anti-CD146 (Becton-Dickinson Biosciences, Shanghai, China). For isolation of CMCs, PE-conjugated anti-Troponin T (cTnT; Becton-Dickinson Biosciences) was used in flow cytometry. Assessment of apoptosis was labeled with a Dead Cell Apoptosis Kit with Annexin V FITC and propidium iodide (PI) (Invitrogen; V13242), and analyzed by flow cytometry. Both pro-apoptotic cells and apoptotic cells were counted. Assessment of proliferation was done with a BrdU Staining Kit for Flow Cytometry (Thermo Fisher Scientific, Inc., Waltham, MA, USA; 8811-6600-42). Reactive oxygen species (ROS) levels were measured in live cells with a dichlorodihydrofluorescein diacetate (DCFH-DA) method that converts 2’,7’-dichlorofluorescin diacetate (DCFDA) to oxidized 2’, 7’ -dichlorofluorescein (DCF), which is detected by by flow cytometry. Flow cytometry data were analyzed and presented with FlowJo software (Flowjo LLC, Ashland, OR, USA).

Undifferentiated MSCs were subjected to flow cytometry analysis to confirm that the hBM-, hAT-, and hWJ-MSCs maintained their immunophenotypic characteristics after growth in the culture. After the 3^rd passage, stem cells were harvested and re-suspended in their own culture medium at a concentration of 1x10⁶ cells/mL. Flow cytometry was performed using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA). The data were analyzed using CellQuest software (Becton Dickinson, San Jose, CA, USA). Debris and dead cells were gated out by forward- and side-scatter profiles. Immunophenotyping of the MSCs was performed with antibodies against the following human antigens: CD3, CD4, CD5, CD7, CD8, CD10, CD11b, CD13, CD14, CD15, CD19, CD29, CD33, CD44, CD45, CD71, CD73, CD90, CD106, CD123, CD146, CD166, HLA-DR, HLA-A, HLA-B, HLA-C, and HLA-G (Becton Dickinson, San Jose, CA, USA).

MSCs were obtained from mucosa of a 40-year old healthy male donor. Cells were cultured in low glucose DMEM (StemCell, USA) supplemented with L-glutamin, penicillin/streptomycin and 20% fetal bovine serum (StemCell, USA) at a concentration of 0.3 × 10⁶ per flask with filter ventilated caps (25 cm²) in a humidified atmosphere of 5% CO₂ and 37°C. MSCs were subcultured every 7 days up to passage 22.
For immunophenotypic characterization, cells were stained with the panels of antibodies against the following surface markers: CD3, CD13, CD14, CD19, CD25, CD29, CD31, CD34, CD38, CD44, CD45, CD69, CD73, CD90, CD105, CD106, CD166 and HLA-DR (Becton Dickinson, USA). The expression of the surface markers was then analyzed using a BD FACS Canto II (Becton Dickinson Bioscience, USA) flow cytometer. The resulting expression profiles revealed high expression levels (>60% positive cells) for CD90, CD105, CD166, CD44, CD73, medium levels (30-60%) for CD13, CD29 and CD69, and very low levels (<5%) for CD45, CD34, CD133, CD3, CD19, CD25, CD38, CD45, CD106, CD31 markers. This immunophenotype was consistent with the reported immunophenotype for MSCs [47 (link)] and did not change in the course of the experiment.