4.5. Transduction by Spinfection in Cultured Cells

MCF-7, A431 and HFF-1 cells were used for stable expression of different vimentin constructs (Figure 1B–D). Cells were seeded at a density of 50,000 cells in T25 culture flask in complete DMEM. The viral supernatant was thawed on ice (occasionally diluted 1:1 with complete DMEM) and polybrene (5 µg/mL) was added for charge neutralization for 15–20 min. Before transduction, the cells were conditioned with complete DMEM containing polybrene for 15–20 min at room temperature. The medium was then replaced with the polybrene/virus supernatant mixture. The culture flasks were spun at 1000 rpm at 32 °C for 1 h and then incubated at 32 °C for 24 h. Cells were then cultured in fresh complete DMEM at 37 °C for 2 days to ensure viral integration and protein expression. These cells were then selected with puromycin 2 µg/mL or hygromycin 300 µg/mL. All the stable cell lines made by retroviral transduction followed by drug selection used in this study are listed in Table 3.

List of stably transduced cell lines used in this study.