Isolated LDs were analyzed and quantified using a fluorescence microscope. In addition, direct samples were taken from the cell cultures to determine and quantify LDs within cells (LDs/cell). For that purpose, the lipophilic TopFluor® Cholesterol or Bodipy Cholesterol dye [23-(dipyrrometheneboron difluoride)-24-norcholesterol] (Avanti® Polar Lipids, AL, United States) was used for selective labeling of LDs. The dye was first dissolved in dimethyl sulfoxide (DMSO) at a concentration of 1 mg/mL. Then, as in Zhou et al. (2019) (link), 2 or 3 drops (300 mL approx.) of TopFluor® Cholesterol were added to 1 mL of culture medium to obtain a final concentration of 10 mM. The solution was stirred and left for 10 min in the dark at room temperature. Finally, the supernatant was removed and washed with distilled water before observation.
The freshly stained LDs were observed under a fluorescence microscope using a 10X ocular and 10X objective lens (Leica DMIRE2, Leica Microsystems Inc., Wetzlar, Germany) coupled to a camera (Leica DFC360-FX) with red and green fluorescence filters (excitation, 545 and 480 nm; emission, 620 and 535 nm, respectively), exposure of 1.233 s and gain 1.55. Isolated LDs and LDs/cell were quantified using the Java-based image processing program imageJ and were expressed as number of LDs or LDs/cell per area (LDs/cm2 and LDs/cell/cm2, respectively).
The freshly stained LDs were observed under a fluorescence microscope using a 10X ocular and 10X objective lens (Leica DMIRE2, Leica Microsystems Inc., Wetzlar, Germany) coupled to a camera (Leica DFC360-FX) with red and green fluorescence filters (excitation, 545 and 480 nm; emission, 620 and 535 nm, respectively), exposure of 1.233 s and gain 1.55. Isolated LDs and LDs/cell were quantified using the Java-based image processing program imageJ and were expressed as number of LDs or LDs/cell per area (LDs/cm2 and LDs/cell/cm2, respectively).
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