Igg2a apc

CD73, CD34, CD90, CD14, CD45, CD31, CD105, class I-HLA and HLA-DR, (Beckman Coulter, IL, Milan, Italy) were used for standard MSC characterization, as previously described [17 ]. Analysis of cell populations was performed with a FACS Navios flow-cytometer (BC).
For stemness marker expression, cells were collected, washed with PBS, fixed and permeabilized where necessary using a Fix and Perm solution (BD Biosciences) for 20 min at room temperature. After washing, samples were stained with primary antibodies against SOX2-AlexaFluor488 (245,610, mouse IgG2a, BD), Nanog-Alexa Fluor488 (N31–355, mouse IgG1k, BD), Oct-3/4-PE (40/OCT3, mouse IgG1k, BD), CD117-PE (104D2, mouse, IgG1k, BD), O4-PE (O4, mouse, IgGM, R&D systems), and corresponding isotype matched controls against CD3 Alexa Fluor 488 (UCTHT1, IgG1k, BD Biosciences), CD4-PE (L200, IgG1k, BD Biosciences) IgM-PE (Goat Anti-Mouse, R&D systems), IgG2A-APC (Mouse, R&D systems). Data were analyzed using AccuriC6 (BD).
Cell cycle analysis was performed on NB-MSCs and BM-MSCs, using 50 μg/ml propidium iodide (Sigma-Aldrich) in 0.1% sodium citrate (Sigma-Aldrich), 0.1% Triton X-100, 10 μg/ml RNAse (Sigma-Aldrich) containing-buffer, after overnight incubation at 4 °C and measured by flow cytometry. Data were analyzed with FlowJo software (Tree Star).

BCSphCs and BC cells were washed in PBS twice, stained for 1 h at 4 °C with conjugated antibodies for CD44 (G44-26, mouse IgG2b; BD Biosciences) and CD24 (ML5, mouse IgG2a; R&D Systems) or corresponding isotype-matched controls IgG2b-PE (BD Biosciences) and IgG2A-APC (R&D Systems) and analyzed by using Accuri C6 (BD Biosciences) flow cytometer. To measure cellular DNA content, cells were washed with PBS and incubated with Nicoletti Buffer for 16 h at 4 °C. Enrichment of CD44^high/CD24^low and CD44^high/CD24^high and OFP-positive subpopulations were accomplished by FACSMelody cell sorter. Collected cells were resuspended in PBS with 2% BSA and 2 mM EDTA and filtered with 70 µm mesh. To verify the purity of the obtained subpopulations we performed a post sorting acquisition. Dead cell exclusion was performed by adding 7-AAD (BD Biosciences) or propidium iodide (PI) (Sigma-Aldrich) to cell staining.