Oil red o stock solution

HUVEC were seeded to detect lipids as well as to perform the MTT assay under the same experimental conditions. Oil Red O Staining was used to detect neutral lipids. After the 24h treatments, the cells were washed three times with PBS, fixed in PFA 10% for 30 min at room temperature, washed once again with PBS, and then stained with 60% filtered Oil Red O stock solution (Sigma-Aldrich) for 20 min. After extensive washing, the Oil Red O was solubilized in 100% isopropanol, was quantified by measuring the absorbance at 500 nm, and was normalized to the cell number by MTT assay [24 (link),25 (link)] after image acquisition using FLoid Cell Imaging Station (Thermo Fisher Scientific). To further confirm the results, staining with BODIPY 493/503 was performed (see dataverse at the following link https://dataverse.unimi.it/dataverse/biomedicines/ accessed on 30 September 2021). The results are the mean of three independent experiments performed in triplicate ± SD.

Staining was performed as previously described with minor modifications (63 (link)). In brief, 4-day-old worms fed OP50 or FC for 4 days were washed three times with M9 buffer. The samples were fixed in 50% isopropanol in phosphate-buffered saline (PBS) for 15 min on ice. Oil red O stock solution (0.5 g/100 mL in isopropanol; Sigma-Aldrich, St. Louis, MO, USA) was diluted in distilled water (dH₂O) to 60% working solution and filtered using a 0.2-μm membrane. Fixed worms were incubated in working solution at 25°C for 20 min. Stained worms were washed with M9 buffer containing 0.5% Triton X-100 and mounted on glass slides using M9 buffer plus 0.5% Triton X-100 for imaging using an BX53 microscope equipped with a DP74 color camera (Olympus, Tokyo, Japan). The measured dye values were normalized to density values determined for the body size, as described above. Next, the dye values measured using ImageQuant TL version 8 (GE Healthcare, Chicago, IL, USA) and Adobe Photoshop Elements were normalized to density values per mm² of a worm’s projection area. Two biological replicates were analyzed for this study.

The Oil Red O Stock Solution (Sigma, Germany) was prepared by dissolving 0,5 g with 100 ml isopropanol protected from light. Cells were washed with 1 × PBS (AppliChem, Germany) and fixed with 4% PFA (Merck, Germany) for 15 min. Slides were covered with cells and 1 × PBS and sealed with parafilm for storage at 4 °C. Next, the slides were covered with Oil Red O working solution for 11 min. The OIL Red O solution was aspirated, the slides were washed afterwards and then covered with haematoxylin (Merck, Germany) for 10–30 s. After another washing step with 1 × PBS, the slides were rinsed with distilled water and covered with Aquatex (Merck, Germany).

HUVEC were seeded in 24-well plates (Greiner bio-one, Frickenhausen, Germany) and cultured in different concentrations of Mg for 24 h. At the end of the experiment, cells were washed three times with PBS, fixed in PFA 10% for 30 min at room temperature, washed once again with PBS and then stained with 60% filtered Oil Red O stock solution (Sigma Aldrich) for 20 min. After extensive washing, Oil Red O was solubilized in 100% isopropanol and quantified by measuring the absorbance at 500 nm. The results are the mean of three independent experiments performed in triplicate.

Fresh liver tissue was frozen in optimal cutting temperature compound (Sakura Finetek, Torrance, CA, USA) and cryostat sections (7 µm-thick) were fixed in ice-cold 10% formalin for 10 min. Fresh oil-red O solution was prepared by dilution of the oil-red O stock solution (Sigma, 3.5 g/l in 99.5% isopropyl alcohol) in distilled water to a final concentration of 0.2%. This solution was mixed thoroughly and filtered through a 0.2-µm filter before use. Next, slides were placed in absolute isopropyl alcohol for 2 min. Subsequently, the sections were stained in pre-warmed oil-red O solution for 20 min in an oven at 60°C, rinsed in distilled water and counterstained with haematoxylin, followed by washing with running tap water. The slides were mounted with Clear Mount™ Mounting solution (Invitrogen, Eugene) and examined using an Olympus BX61 microscope.

The cells (1 × 10⁶) were seeded on glass cover slips in 6-well plates and incubated for 24 h at 37 °C in a humidified CO₂ incubator. The cells were then fixed using 4% formaldehyde (PFA) made in PBS at room temperature for 5 min, and washed once using 60% isopropyl alcohol for 5 min. Oil Red-O stock solution (0.25%) (Sigma-Aldrich, St. Louis, MO, USA) was prepared in 20 mL of isopropyl alcohol and diluted in distilled water and filtered before use. The diluted Oil Red-O solution was used to stain the cells at room temperature for 20 min, followed by rinsing with distilled water, and followed by staining with hematoxylin (Sigma-Aldrich) to visualize nuclei. The cells were imaged on a Keyence BZ-X800 fluorescence microscope (Keyence Corp., Osaka, Japan). A total of four fields of each condition were used for the quantification of the Oil red-O staining using the NIH ImageJ software.

To investigate lipid accumulation in worms, we performed staining using a previously established method with slight modifications [22 (link)]. We examined lipid accumulation in worms of various experimental ages, including four-, five-, six-, and seven-day-old worms subjected to different feeding strategies (OP50, OP50 + LL100933, OP50 + LL12007, LL100933, and LL12007 feeding). Briefly, worms fed different feeds were washed thrice with M9 buffer. The specimens were immobilized in 50% isopropanol solution in phosphate-buffered saline (PBS) for 15 min on ice. The Oil Red O stock solution (0.5 g per 100 mL in isopropanol; Sigma-Aldrich, St. Louis, MO, USA) was diluted with distilled water (dH₂O) to create a 60% working solution, which was then filtered through a 0.2 μm membrane filter. Immobile worms were placed in a functional solution and incubated at 25 °C for 20 min. After staining, immobilized worms were rinsed with M9 buffer containing 0.5% Triton X-100 and placed on glass slides for imaging. Worms were randomly selected for observation [23 (link)]. Imaging was performed using a BX53 microscope fitted with a DP73 color camera (Olympus, Tokyo, Japan). The dye intensity was then measured using the ImageJ software (v1.53), as described previously [24 (link)].