Cell Sorting

To certify that the GFP has been at least partially integrated, the cells are viewed through a confocal microscope, which works by shining lasers at cells. This does not provide any data but simply allowed the visualization of how the cells looked with the genome modification.
After modifying the plasmids and transfecting them into the 4T1 cells, the cells were placed into a cell sorter (Figure 4). The cell sorter works by shooting 395nm light at the cells. The cells that fluoresce back green have positively integrated the modified plasmid, which contains the GFP. The cell sorter will then calculate the concentration of cells that are green. After the data is transferred to the computer, the cell sorter separates the cell population into positively and negatively integrated. The cell sorter keeps the cells alive, and then the modified cells can eventually be reinserted into a balb/c mouse; however, prior to that, new cell cultures of the modified cells need to be grown, and the IL-22 knock-out needs to be verified.
The cell sorter is able to separate colonies by color: the green cell colonies that are most likely positive for the modification are transferred into 96 well plates. In those wells the 4T1 cells grow to confluence as a homogenous population, ensuring that all the cells in that well are modified and the plasmid was successfully transfected. Thus, the cells can be properly identified through flow cytometry.
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