Figure 5.

Mesenchymal stem cells were efficiently transfected in the presence of DOPE/CHEMS and HDACi. (A) MSCs were transfected with Rho-pDNA polyplexes (complexation of 350 ng Rho-pDNA at the ratio of 1 μg pDNA to 2 μl Turbofect) using low speed centrifugation. Four hour post-transfection, the media was replaced with 0.4% trypan blue/1× PBS. Cell images were taken before (–) and after (+) quenching with 0.4% trypan blue. Bar represents 200 μm. (B) Polyplex of Turbofect/pDNA (350 ng of pDNA) was added to MSC and centrifuged at 280 g (5 min). At various time points, post-transfection, cells were treated with pAA/DNAse to effectively remove extracellular pDNA. Following trypsinization, cells were collected for pAA/urea lysis buffer treatment. The absolute copy numbers of EGFP DNA was quantified using the standards by real-time qPCR. Bar graph shows Log₁₀ mean and ± SEM (n = 4). (C) MSC was transfected with Turbofect/pDNA in the presence or absence of DOPE/CHEMS plus 10 μM Tubastatin A. After twenty four hours incubation, transfection efficiency (percentage of EGFP+ cells normalized to the total number of cells) was acquired using FACS. Results were presented as mean ± SD (n = 3). Significant differences between data points were calculated using two tailed Student's t-test. **P < 0.005.