One of the first things to identify in flow cytometry is your cell population. This can be done by its forward and side scatter characteristics or using side scatter plus a marker such as CD45 for cell population identification. After this you need to determine the location of the negative population. To do this you should always use an unstained sample. This will allow you to determine the level of background fluorescence or autofluorescence and set your voltages and negative gates appropriately.
Fig. 19. Unstained controls. Unstained lymphocytes (A) are used to determine the background autofluorescence to set the negative population allowing cells stained with CD4 (MCA1267A647) and CD8 (MCA1226F) to be visualized (B).