Bodipy 493 504

For LD staining in L. incisa cells 10 ml of samples were collected at selected time points of nitrogen deprivation, pelleted by centrifugation at 1000 g for 5 min and fixed with 4% (w/v) paraformaldehyde in 0.1 M PBS, pH 7.4, overnight at 4 °C. Fixed samples were washed three times with the same buffer and stained with 10 μg/ml Bodipy 493/504 (Thermo Fisher Scientific) diluted in 0.1 M PBS buffer, pH 7.4, for 1 h at RT with gentle agitation. Then samples were washed 3 times with the PBS buffer only and re-suspended in Prolong gold anti-fade reagent (Thermo Fisher Scientific). LD staining in yeast cells was performed as described in [40 (link)].

Cells were fixed in a mixture of 4% (w/v) paraformaldehyde in PBS (pH 7.4) overnight at 4 °C. After three washes in PBS buffer, cells were incubated with Bodipy 493/504 (Thermo Scientific, Grand Island, USA) at a final concentration of 10 µg/ml in PBS buffer for 1 h at room temperature. This was followed by the washing of cells three times in the same buffer and re-suspended in ProLong Gold anti-fade reagent (Thermo Fischer Scientific, Waltham, USA). Samples were then analyzed with a Zeiss LSM 510 META confocal laser scanning microscope (Carl Zeiss, Jena, Germany) using a 63× Plan-Apochromat 1.4 NA oil-immersion lens. An argon laser was used for Bodipy 493/504 and chlorophyll excitation and the emission spectra were collected in two separate channels, 500–515 nm and 630–670 nm, respectively. Z-series images were collected and processed with the LSM 5 Image Browser (Carl Zeiss, Jena, Germany). LD morphometrics were performed by the same software using 3D reconstruction confocal images of 70 cells for each analyzed line.