Figure 2. Using FACS to rapidly isolate cells with inactivated GDP-fucose transporter. Cells transfected with constructs expressing the ZFNs were cultured to near confluence and subcultured at 1:6 ratio for 2 passages. Ten million of the resulting cells were labeled with biotinylated AAL and Cy3-conjugated streptavidin. Stained cells were sorted on a Becton Dickenson FACSAria IIu SORP cell sorter. (A) FACS histogram for transfected cells at the first round of sorting. The sorting gate for AAL-negative (AAL-ve) cells was set to collect the lowest 0.5% of AAL-stained cells. Approximately 12,000 cells were collected from a total of 3.5 million cells sorted. These cells were cultured for 2 weeks before being subjected to a second round of sorting. (B) Second round of FACS shows that more than 30% of the cells are AAL-ve cells after the first round of sorting. Single AAL-ve cells were isolated from this pool for further characterization.