Propidium iodide staining and imaging

To evaluate the amount of dead CaGFP, CgGFP or SaGFP cells in a fresh induction culture, microorganisms were stained with 2.5 ng/ml of propidium iodide (Sigma-Aldrich). Propidium iodide (PI) is widely used as a vital dye that is not permeant to live cells, but labels the nucleus in dying cells, which lack an intact plasma membrane. Thimerosal-killed Cell Tracker^™Green-labeled C. albicans or C. glabrata and ethanol-killed Cell Tracker^™Green-labeled S. aureus cells, respectively, served as controls for a positive PI staining. Fluorescence images of C. albicans and C. glabrata samples were acquired with the Zeiss AxioObserver Z.1 microscope using a 40x objective (EC Plan-Neofluar 40x/0,75) and processed using the ZEN 2.3 software (Carl Zeiss). Images of S. aureus samples were acquired with the LSM 780 confocal microscope using a 63x objective (Plan-Apochromat 63x/1.4 Oil DIC) and processed using the ZEN 2.3 software.