Dulbecco modified eagle medium (dmem)

Rat C6 glioma cells47 (link) were cultured in Dulbecco’s modified Eagle’s medium (High Glucose) (D-MEM, 044–29765, Wako, Tokyo, Japan) supplemented with 10% foetal bovine serum (172012; Sigma-Aldrich) and 1% penicillin/streptomycin (168–23191; Wako) and maintained in 5% CO₂ at 37 °C. The cells were plated at a density of 1.0 × 10⁵ cells per well on a coverslip sheet (Cell Desk LF, MS-92132; Sumitomo Bakelite, Tokyo, Japan) for 2 days. For glucose deprivation, the cells were incubated in D-MEM (no glucose) (042–32255, Wako) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin for 3 days. The cells were immunostained for NSE with essentially a similar protocol as used for cerebellar slices. The NSE immunofluorescence images were captured using a confocal microscope and the same settings from 10 randomly selected regions from 2 independent cultures (5 regions from one culture) in each experimental group. The fluorescence intensity was measured using ImageJ. The background intensity was subtracted from the fluorescence intensity. The averaged fluorescence intensity of control cells was taken as 100%, and the relative values were calculated.

The HCE-T human corneal epithelial cell line (RIKEN CELL BANK, Ibaraki, Japan) derived from human corneal epithelial cells was maintained in DMEM (Wako Pure Chemical Industries, Ltd., Osaka, Japan):HamF12 (Wako Pure Chemical Industries, Ltd.) (1:1) medium supplemented with 5% fetal bovine serum (FBS), insulin (Sigma-Aldrich, St. Louis, MO, USA) at a final concentration of 5 μg/mL, and human epidermal growth factor (Roche, Basel, Switzerland) at a final concentration of 10 ng/mL. This cell line was kindly supplied by Masao Taki, Tokyo Metropolitan University. The SRA01/04 human lens epithelial cell line (RIKEN CELL BANK) derived from human lens epithelial cells was maintained in DMEM (Wako Pure Chemical Industries, Ltd.) medium supplemented with 20% FBS. This cell line was kindly supplied by Hiroshi Sasaki, Kanazawa Medical University. The cells were seeded onto 10 cm dishes (Asahi Glass, Tokyo, Japan) at a density of 1 × 10⁶ cells/mL with total volume of 11.6 mL medium to adjust the medium layer thickness to 2 mm for ideal exposure. After overnight culturing, the cells were exposed to 60 GHz. The cells were collected 24 h after exposure to millimeter-wavelength radiation. For positive controls, cells were treated with 10 μg/mL bleomycin for 1 h for the genotoxicity test and with heat (43 °C for 2 h and then 37 °C for 1 or 2 h) for the Hsp expression test.