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For identification of the phenotype of AF-MSCs from passages 4-5, cells were collected by centrifugation at 1,200 rpm for 6 min, washed once in phosphate buffered saline (PBS) with 0.2% fetal calf serum (FCS), and centrifuged again. A total of 5 × 10⁵ cells were resuspended in 50 μL of PBS with 1% BSA and incubated with fluorescein isothiocyanate- (FITC-) conjugated mouse anti-human antibodies against CD44 (Invitrogen), CD34 (Miltenyi Biotech), CD90 (Molecular Probes, Life technologies) or phycoerythrin- (PE-) labelled CD105 (Invitrogen), and appropriate isotype control mouse IgG2A-FITC (Miltenyi Biotec) or IgG1-PE (Molecular Probes, Life Technologies). Samples were incubated in the dark at 4°C for 30 min and analysis was performed on a flow cytometer BD FACSCanto II (Beckton and Dickinson) with BD FACSDiva software.

Flow cytometry analysis of trypsin-dissociated cultures was performed using antibodies for CD45-FITC, CD34-APC, and CD38-PE and corresponding isotype controls IgG2a-FITC, IgG2a-APC, and IgG2a-PE, all from Miltenyi and used as per the manufacturer's instructions. Cells were enumerated using fluorospheres (Beckman Coulter). Hematopoietic cells were assessed based on positive CD45 surface expression and progenitor cells were quantified based on positive CD34 surface expression and lack of CD38 expression. For gating strategy, see Supplementary Figures S1 and S2 (Supplementary Data are available online at www.liebertpub.com/tec).
For 3D imaging of coculture spheroids, cells were prepared by first labeling MSCs with Cell Tracker^™ Red CMTPX (Molecular Probes) and CD34⁺ cells with Green CellTrace^™ CFSE (Molecular Probes), as previously described.^{19 (link)} Following 7 days of coculture, spheroids were fixed, washed of detached cells using a cell strainer, and imaged on a Leica TCS SP5 confocal microscope.

For identification of the phenotype of AF-MSCs from passages 4-5, cells were collected by centrifugation at 1,200 rpm for 6 min, washed once in phosphate buffered saline (PBS) with 0.2% fetal calf serum (FCS), and centrifuged again. A total of 5 × 10⁵ cells were resuspended in 50 μL of PBS with 1% BSA and incubated with fluorescein isothiocyanate- (FITC-) conjugated mouse anti-human antibodies against CD44 (Invitrogen), CD34 (Miltenyi Biotech), CD90 (Molecular Probes, Life Technologies), or phycoerythrin- (PE-) labeled CD105 (Invitrogen) and appropriate isotype control—mouse IgG2A-FITC (Miltenyi Biotech) or IgG1-PE (Molecular Probes, Life Technologies). Samples were incubated in the dark at 4°C for 30 min and finally analyzed with the Millipore Guava® easyCyte 8HT flow cytometer, using the InCyte 2.2.2 software. Ten thousand events were collected for each sample.