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 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.
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