Chapter 4 —— 80 —— combinations at the indicated MOIs. Gene editing experiments were assessed by reporter-directed flow cytometry at early and late timepoints posttransduction for quantifying transient and stable transduction levels, respectively. Gene editing experiments in HeLa cells were assessed at 3 days and 14 days post-transduction and in hMSCs and human myoblasts were examined at 3 days and 20 days post-transduction. Additional gene editing control experiments in HeLa cells were assessed at 3-, 7-, 10-, 14- and 17days post-transduction. These extra control experiments included the use of AAV-HRS1 and AAV-HRS1GS1 at 2 TU cell-1 and AdVP.C9KARA and AdVP.mCherry [25] at 0, 5 or 20 GC cell-1. Live-cell microscopy analyses The live-cell imaging of cells expressing reporter proteins at the indicated early and late timepoints post-transduction was done with the aid of an AF6000 LX inverted fluorescence microscope (Leica). The acquired images were examined with the aid of LAS X (Leica Microsystems). The monitoring of hMSCs transduced with second-generation AdV versus AdVP vectors was done in an Incucyte S3 live-cell imaging analysis system (Sartorius) and realtime analyzed by using the Incucyte software (Essen BioScience). Flow cytometry The quantification of cell transduction efficiencies and stable transduction efficiencies as well as mean fluorescence intensities was performed on a per sample basis by using a BD LSR II flow cytometer (BD Biosciences). In brief, cells were washed with PBS (pH 7.4) and, after treatments with a 0.05% trypsin-EDTA solution (Thermo Fisher Scientific; cat. no.: 15400–054), cells present in the resulting cell suspensions were collected by a brief centrifugation and resuspended in FACS buffer consisting of PBS (pH 7.4) supplemented with 0.5% (w/v) bovine serum albumin and 2 mM EDTA (pH 8.0). Mock-transduced cells treated in parallel, were used to establish the background fluorescence threshold levels. For each sample, at least 10 000
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