Nile red solution

Normal and GO-derived OFs (5 × 10³/cm²) were cultured in a six-well plate and incubated in serum-free DMEM/F12 supplemented with 33 μM biotin, 17 μM pantothenic acid, 0.2 nM triiodothyronine (T₃), 10 μg/mL transferrin, 0.2 μM carbaprostacyclin (cPGI₂; Cayman Chemical, Ann Arbor, MI, USA), 0.1 mM isobutylmethylxanthine (IBMX), 1 μM dexamethasone, and 1 μM insulin (Sigma-Aldrich). The differentiation-induced medium was replaced every day for 4 days. The medium exchanged to a maturation medium without 1 μM dexamethasone and 0.1 mM IBMX (all from Sigma-Aldrich) for 6 days and was replaced every other day. Lipid droplets were stained using 0.5 μg/mL Nile red solution (Sigma-Aldrich).

Larval brains were dissected at 120 h AEL and fixed in 4% of PFA/PBS at room temperature for 30 min, and washed in PBST (PBS, 3% TritonX) three times for 20 min each time. Fixed brains were incubated in the Nile Red solution (Sigma Aldrich, 19123, diluted 1:100 in PBST from a 100 mg/ml stock solution in acetone) at 4 °C overnight and then washed in PBST for 30 min each time. Larval brains were mounted on glass slides with the anti-fade mounting solution and imaged with a Zeiss LSM700 upright confocal microscope.

Cells or liver sections were fixed with 4% formaldehyde in PBS, and stained with 0.5% Oil Red O (Sigma) in propylene glycol^{67 (link)}. Excessive staining was removed by wash with 70% ethanol, and stained cells were photographed. For Nile red staining, 0.05 mg/mL Nile red solution (Sigma) was used to visualize lipid droplets.

Neutral lipids were quantitatively measured using the procedure published by Chen et al. [55 (link)]. Chlorella cells grown under nutrient-limited condition were diluted to OD₇₅₀ = 0.06, and 5 μL samples were introduced into the individual wells of a 96-microplate containing 3 μL of a 50 μg/mL Nile red solution (Sigma Aldrich, St. Louis, MO, United States). Then, 292 μL of an aqueous solution containing 25% dimethyl sulfoxide (Sigma Aldrich, St. Louis, MO, United States) was added. The 96-well plate was vortexed (120 rpm) and incubated at 40°C for 10 minutes. After the algal cells were stained, the fluorescence emission was recorded using a Varian spectrophotometer (Thermo Fisher Scientific, Rockford, IL, USA) equipped with a 96-well plate reader. Excitation and emission wavelengths of 530 nm and 575 nm, respectively, were selected. Eight replicates of each treatment were analyzed. Images were acquired using a Zeiss Cell Observer SD (Carl Zeiss Microscopy GmbH, Jena, Germany).

For Nile red staining, cells were washed in PBS and fixed with 4% formaldehyde at room temperature for 15 min. Fixed cells were stained with 1 μg/mL Nile red solution (Sigma, St. Louis, MO, USA) for 15 min. Images of stained cells were acquired using confocal microscopy. Fluorescence intensity was quantified in the Image J software and normalized by the number of cells.

The plates were processed for measurements of triglyceride accumulation and DNA content, as described previously [48 (link),49 (link),50 (link),51 (link),52 (link)]. Briefly, the cells were rinsed with Dulbecco’s phosphate-buffered saline (DPBS) and then treated with 200 μL/well of a dye mixture: ~19 mL DPBS, 20 drops/mL NucBlue^® Live ReadyProbes^® Reagent (DNA content; Thermo cat # R37605) and 500 μL Nile Red solution (40 μg/mL solution; Sigma cat #72485-100MG). After the addition, the plates were protected from light and incubated for forty minutes, and then the fluorescence was measured using a Molecular Devices SpectraMax iD5 (San Jose, CA) at 485/572 nm excitation/emission for Nile Red and 360/460 for NucBlue. Triglyceride accumulation was reported as percent activity, corrected for intra-assay differentiated vehicle control responses and relative to the rosiglitazone-induced maximum. The DNA content was reported as percent activity relative to the differentiated vehicle control responses. Normalized triglyceride content was calculated as total triglycerides per well per unit DNA content (used as a proxy for triglycerides per cell). Four technical replicates (wells within each assay plate) and three biological replicates (separate cell passages/assays) were utilized for every test chemical and concentration.