Chapter 4 —— 81 —— viable single cells were acquired and subsequently analyzed with the aid of the FlowJo 10.9.0 software (BD Biosciences). Cell viability assays hMSCs were seeded at a density of 4 × 103 cells per well of 96-well plates (Greiner-BioOne) and, 16–18 h later, they were exposed for 24 h to secondgeneration vector AdV.Δ2.Cas9 or to high-capacity vector AdVP.C9KARA at different MOIs. Mock- and vector-transduced hMSCs were then kept in an Incucyte S3 apparatus during 4 days for live-cell real-time monitoring of cell confluency and division rates. The Incucyte software (Essen BioScience) was used for this purpose (https://figshare.com/s/a999fdb4400aaf1828c5). Moreover, at consecutive 4 days post-transduction, the metabolic activities of hMSCs were determined by colorimetric quantification of the conversion of the tetrazolium salt (WST-8) to formazan using the CCK-8 kit (Boster Biological Technology; cat. no.: AR1160). In brief, after substituting regular culture medium by 100 μl of medium containing 10 μl of CCK-8 solution, hMSCs were incubated for 1 h at 37℃. Next, the absorbance of the solution in each well, directly proportional to the amounts of viable metabolically active cells, was measured at 450 nm in a multimode plate reader (PerkinElmer VICTOR X3). Cell differentiation assays Myogenic differentiation was initiated by exposing to low-mitogen medium confluent cultures of unmodified myoblasts and of DMD edited myoblasts seeded in plates pre-coated with 0.1% (w/v) gelatin. This differentiation medium consisted of phenol red-free DMEM (Thermo Fisher Scientific; cat. no.: 11880–028) supplemented with 100 U ml-1 penicillin/streptomycin, 10 μg ml-1 human insulin (Sigma-Aldrich; cat. no.: 19278) and 100 μg ml-1 human holo-transferrin (Sigma-Aldrich; cat. no.: T0665). After 4–5 days under differentiation conditions, total RNA was isolated from the resulting syncytial myotubes for multiplex reverse transcriptase-polymerase chain
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