P2307

Nile red staining was performed following the protocol described by Greenspan et al. with modifications [78 ]. In brief, nile red stock solution of 1 mg/mL concentration was obtained by dissolving 5 mg of nile red powder (Sigma-Aldrich N3013, USA) in 5 mL of acetone. The stock solution was diluted to 1:100 nile red staining solution in 1 mM trizma-maleate (Sigma-Aldrich T3128, USA) and 3% w/v Polyvinylpyrrolidone (Sigma-Aldrich P2307, USA). The differentiated adipocytes were washed with PBS (Sigma-Aldrich D8537, USA) and fixed with 4% paraformaldehyde for 1 h. Then, the cells were stained with nile red to visualize the lipid droplets and DAPI (Life Technologies P36930, USA) to stain the nucleus. Olympus fluorescent microscope was used to observe the stained cells and imaged using cellSens Standard software.

Nile red stain was prepared following the protocol of Greenspan et al. [60 (link)] with minor modifications. Nile red was prepared by dissolving 5 mg Nile red powder (Sigma-Aldrich N3013, USA) in 5 mL of acetone to obtain 1 mg/mL stock solution. Nile red stock solution was diluted in 1mM trizma-maleate (Sigma-Aldrich T3128, USA) and 3% w/v Polyvinylpyrrolidone (Sigma-Aldrich P2307, USA) to obtain 1:100 Nile red stain solution. Differentiation media was discarded, the adipocytes cells were washed with PBS and fixed with 4% paraformaldehyde for 1 h. Fixed cells were stained directly with 1:100 Nile red stain solution and DAPI stain (Life Technologies P36930, Eugene, OR, USA). Stained cells were imaged under the Olympus fluorescent microscope using Cellsens standard software.