Figure 3. FACS sorting parameters for selecting eGFP-positive S2 cells.

The cell selection was performed with BD FACSAria II. Laser extension was set at 2 and FSC Area scaling at 0.6. (A) The first sorting gate was set with FSC-A on X-axis and SSC-A on Y-axis, both with linear scale, to select for the particles that have the size of S2 cells and normal internal complexity. Wild-type S2 cells were used to determine the gate and kept for the following experiments. (B) The second gate selects for singlets which uses SSC-H as the X-axis and SSC-W as the Y-axis, both with linear scale. High SSC-W population was discarded. (C) The final gate was set on log scaled GFP signal intensity and SSC-A was simply included for easier data visualization. The cutoff GFP intensity was determined by analyzing wild-type S2 cells to ensure that the threshold is right above the maximum auto-fluorescence from S2 cells. The P1–4 population was the only cell population collected for the following experiments. These criteria select about 1% of the sorted S2 cells, but the actual number varies from experiments due to transfection efficiency fluctuation. Specific parameters may be examined and altered when setting up the experiment for the first time, and the sorting effectiveness should always be verified before the actual sorting by test sorting of samples and viewing by fluorescence microscopy.