Chapter 5 —— 126 —— transduction, respectively, HeLa cells and hMSCs were cultured in the absence and in the presence of ouabain at final concentrations of 0.2 μM and 1.0 μM, respectively. Parallel cultures of mock-transduced cells were also exposed and not exposed to ouabain. Subsequently, genome modification endpoints were assessed at the indicated timepoints post-transduction by a combination of reporter-directed flow cytometry and AAVS1, ATP1A1, and LMNA genotyping analyses. The latter analysis involved RFLP and junction PCR assays whose details are specified in Supplementary Tables S6 - S9, S12 and S13. Microscopy analysis The direct fluorescence microscopy analysis of reporter-tagged LMNA at early and late timepoints post-transduction was performed with an AF6000 LX inverted fluorescence microscope (Leica) and the resulting images were examined with the aid of the LAS X software (Leica Microsystems). Flow cytometry analysis Selector AAV vector transduction efficiencies and corresponding mean fluorescence intensities per cell were determined through reporter-directed flow cytometry using a BD LSR II FACS (BD Biosciences). The same apparatus was also used to quantify AAV stable transduction levels and LMNA-tagging frequencies. In brief, mock- and vector-transduced cells were rinsed with PBS (pH 7.4) and incubated in 0.05% trypsin-EDTA (Thermo Fisher Scientific; Cat. No.: 15400-054). The resulting cell suspensions were then collected in cultured medium, briefly centrifuged and resuspended in FACS buffer composed of PBS (pH 7.4) containing 0.5% (w/v) BSA and 2 mM EDTA (pH 8.0). The mock-transduced cells served to set the background fluorescence threshold cutoff. At least 10,000 viable single cells were acquired per sample. The resulting datasets were analyzed with the aid of the FlowJo 10.9.0 software (BD Biosciences).
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