Schwann cells were seeded at 2 × 105/mL on poly-l-lysine-coated coverslips. When they reached 70% confluence, the cells were starved overnight and observed in duplicate under the three conditions described above (control, or high glucose with or without quercetin). The cells were treated with 5% bovine serum albumin, followed by primary antibody (dilution 1:100; rabbit anti-Beclin-1 antibody and rabbit anti-LC3 A/B antibody; Abcam, Cambridge, UK) at 4°C overnight. PBS was used in place of primary antibody as the negative control.
Following PBS washes, the cells were incubated with secondary antibody (TRITC-conjugated Affinipure goat anti-rabbit IgG; Beijing Xiya Jinqiao Biotechnology Co., Ltd.) at 37°C for 20 minutes, and stained with fresh DAPI for 10 minutes. The fluorescent intensity of different groups of cells was analyzed using an Olympus FluoView FV 1000 (Olympus Corporation; excitation 364 nm, emission 488 nm for DAPI; excitation 547 nm, emission 620 nm for TRITC). Average absorbance values of each cell were measured by FV10-ASW 3.0 analysis software (Olympus Corporation). Five fields of view were randomly chosen from each group for analysis. The experiment was performed twice.
Following PBS washes, the cells were incubated with secondary antibody (TRITC-conjugated Affinipure goat anti-rabbit IgG; Beijing Xiya Jinqiao Biotechnology Co., Ltd.) at 37°C for 20 minutes, and stained with fresh DAPI for 10 minutes. The fluorescent intensity of different groups of cells was analyzed using an Olympus FluoView FV 1000 (Olympus Corporation; excitation 364 nm, emission 488 nm for DAPI; excitation 547 nm, emission 620 nm for TRITC). Average absorbance values of each cell were measured by FV10-ASW 3.0 analysis software (Olympus Corporation). Five fields of view were randomly chosen from each group for analysis. The experiment was performed twice.