Sytox green

Neutral lipid accumulation was evaluated by flow cytometry using the fluorescent marker BODIPY 493/503 (Thermo Fisher Scientific, USA). Plasma membrane integrity was evaluated using SYTOX Green (Thermo Fisher Scientific, USA), which is impermeable to intact membranes, but penetrates compromised membranes and stains nucleic acids. Untreated and treated cells (obtained as described in “Treatment of fungal cells”) were incubated with 200 μl containing 20 μM of each fluorochrome for 30 min at room temperature in the dark. Cells were washed in PBS, lightly fixed in 2% formaldehyde, and washed again in PBS. The fluorescence intensity of stained cell populations was quantified in a BD Accuri C6 flow cytometer (BD Biosciences, USA) that counted 10,000 or 2000 events per sample for BODIPY 493/503 or SYTOX Green, respectively. Data were analyzed using BD Accuri C6 software. Experiments were repeated three times. Data depicted in the results section shows the mean and standard error of the mean of one representative experiment.

At designated time points, cells were washed with 1 x PBS (1X) and detached with 0.05% Trypsin for 5 min at 37°C. Detached cells were mixed with equal volume of DMEM + 10% FBS, passed through 40 μm strainer and collected by centrifugation (500 x g, 5 min, 4°C). Cells were washed with 1 x PBS (1X) and resuspended in DMEM + 10% FBS supplemented with 10 μg/mL gentamicin. SYTOX Green (ThermoFisher) was supplemented to differentiate dead cells when necessary. Cells were sorted with Aria III (BD) (tagBFP Ex: 405 nm Em: 450/50 nm; Timer⁵¹⁰ and SYTOX Green Ex: 488 nm Em: 530/30 nm; Timer⁵⁸⁰ Ex: 561 nm Em: 586/15 nm; smURFP Ex: 633 nm Em: 660/20 nm) to collect uninfected cells, infected cells with dormant or SPI-2 S. Typhimurium populations. The recorded events were gated according to the strategy described (S2 Fig).

For centrifugal elutriation, cells were inoculated at 1 × 10⁵ cells/mL and grown overnight in 1–2 liters of YEPR at 30°C until 5.10⁷ cells/mL. Cells were harvested by centrifugation and resuspended in 400 ml of cold YEPRaff, and sonicated 30 s on VibraCell 72405 sonicator set at amplitude 60. Cells were kept on ice, loaded onto a Beckman J6-MC centrifuge (JE5.0 rotor) at 3600 rpm, with the pump (Masterflex, Cole Parmer, Barrington, IL) flow rate set at 20 mL/min until the 40 mL chamber was full, and left to equilibrate for 10 min with cold SC-D medium. Pump speed was then gradually increased by 0.02 increments until small daughter cells were elutriated (between 22 and 25 mL/min). Cells were collected until a sufficient amount was reached. For MET3p-CLN2 cells were elutriated in SC-D in which the methionine was omitted and then released in SC-D with or without methionine. For GAL10p-CLN3 cells were elutriated in SC-R and then released in SC-D, SC-R + G00.1% and SC-G. Modal cell volume was measured with a Cell Analyser System (CASY1 TTC, Schärfe System, Reutlingen, Germany). Cell cycle progression was determined by measuring budding index and DNA content using Sytox Green and FacsCalibur cytometer (Becton Dickinson, France).

Samples were prepared for flow cytometry, as described previously [29 (link)], with modifications according to specific strain requirements. Yeast cells were stained using propidium iodide (PI) (16 μg/mL) (Sigma Aldrich, St. Louis, MO, USA) (strains Y1023, Y1024) or SYTOX Green (0.5 µM) (Invitrogen, Carlsbad, CA, USA) (strains Y1006, Y1012, Y1013, Y1014, Y1017, Y1018, Y1019, Y1020). The DNA content was determined by measuring the PI or SYTOX Green fluorescence signal (FL2 or FL1, respectively) using Becton Dickinson FACS Calibur and CellQuest software (BD Bioscience, San Jose, CA, United States).