Metformin Induces Autophagy in FaDu Cells

FaDu cells were grown on glass coverslips in 6-well plates and treated with 0, 5, or 10 mM metformin for 48 h. The treated cells were fixed with 4% formaldehyde for 10 min and permeabilized with 0.1% Triton X-100 in PBS at 4 °C for 10 min. The samples were then blocked with 3% bovine serum albumin (BSA) in PBS at 37 °C for 30 min, then stained with anti-LC3B (Cell Signaling Technology no. 3868; diluted 1:200) and Alexa Fluor 488-conjugated anti-mouse antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA; diluted 1:400). 4′,6-diamidino-2-phenylindole (DAPI) (0.2 μg/mL) was used for nuclear staining. The stained cells were then analyzed under a confocal microscopy (Leica TCS SP8X laser-scanning microscope using a 63 × 1.4 numerical aperture objective) [56 (link)].