Igg2a apc

The primary antibodies used were as follows: MEF2D (BD Biosciences, San Jose, CA, USA); MEF2A (C‐21 Santa Cruz Biotechnology, Dallas, TX, USA); p53 and pERK (Cell Signaling Technology, Leiden, the Netherlands); p21, Actin, anti‐BrdU, FLAG M2 and RACK‐1 (Sigma‐Aldrich); RAS (Abcam, Cambridge, MA, USA); GFP and HDAC4 (Paroni et al., 2004); and HDAC5 (Clocchiatti et al., 2015). Anti‐CD44‐FITC (BD Biosciences) and CD24‐APC (BioLegend, San Diego, CA, USA) were used with matched control antibodies, anti‐mouse IgG1 FITC and IgG2a APC (Miltenyi Biotec, Bergisch Gladbach, Germany). HDAC7 antibodies were generated in rabbits by injecting recombinant histidine‐tagged HDAC7 fragment aa 261–522. For anti‐HDAC7 antibody purification, HDAC7 was fused to glutathione S‐transferase and cross‐linked to glutathione‐Sepharose as described previously (Paroni et al., 2004).

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.