Nile red fluorescent dye

Ten μl of AS from a one-week-old culture suspended in sterile saline solution with 0,02% w/v of Tween 80 and 10 μl of SS from 4-day-old culture was dropped directly onto a glass slide and covered with a coverslip.
Nile red fluorescent dye (1 mg/ml solution in acetone; Sigma-Aldrich, Germany) was used to detect the lipid reserves in the cytoplasm of sporidia. The dye was first dissolved in 1 ml of acetone, then added at the ratio 1/10 (v/v) to the sporidia suspension and incubated in the dark at room temperature for one hour. The sporidia were harvested by centrifugation at 4000 rpm, twice washed with distilled water and re-suspended in a sodium acetate solution buffer (5mM) [31 (link)]. The sporidia samples were observed in white light and red fluorescence microscopy (microscope Zeiss-Axioskop 2 MOT). The microscope was fitted with a camera-type Axiocam HRC color Carl Zeiss technology, operating with the AxioVision 3.1. software. Size and length measurements were performed on 50 cells of each of AS and SS.

Intracellular fatty acids were stained with Nile-red as described [20 (link)]. HCT116 cells incubated in the presence or absence of drug for 48 h were stained with 500 nM Nile-red fluorescent dye (Sigma-Aldrich, St. Louis, MO, USA) in PBS. The fluorescence stained cells were analyzed by an IncuCyte live cell imaging system (Essen BioScience, Ann Arbor, MI, USA).

The intracellular neutral lipid distribution in HCT116 cells was examined by staining the cells with nile red fluorescent dye (Sigma-Aldrich, St. Louis, MO, USA) diluted in Hanks and 20 mM Hepes buffer (HHBS), pH 7. The details are described in the Supplemental Materials. The images were captured with an Olympus FV1000 confocal microscope (Olympus Corporation, Japan) with an excitation wavelength of 486 nm; the emission was measured at 570 nm, following the method of Elsey et al.^{37 (link)}For flow cytometer analysis, cells stained with nile red alone were washed twice with 1 ml of cold FACS buffer and centrifuged at 1 500 r.p.m. at 4 °C for 5 min. All pellets were resuspended with 1 ml PBS + 1% paraformaldehyde and incubated on ice in the dark for 15 min. Cells were washed twice, as described above, and finally resuspended with 1 ml of FACS buffer and detected by a BD FACS (FACSCalibur; Becton Dickinson, Franklin Lakes, NJ, USA).