Dulbecco s modified eagle s minimum essential medium

The LC-HK2 cells (initially called HK2, a cell line established from human non-small cell lung carcinoma) [49 (link)] and hTERT RPE-1 (RPE-1) cells (derived from normal human retina epithelium - ATCC CRL-4000) were cultured in Dulbecco's Modified Eagle's Minimum Essential Medium (Sigma, St Louis, MO, USA), supplemented with 10% fetal bovine serum, in a humidified atmosphere with 5% CO2 at 37°C.

Mouse primary astrocytes were isolated from primary mixed glial-cell cultures of newborn ICR mice, as described previously^{82 (link),83 (link)}. The purity of the astrocytes was >95%, as determined by immunostaining with the anti-GFAP antibody. Astrocytes were plated at a density of 3 × 10⁵ cells/well in 6-well dishes. Cells were maintained in Dulbecco’s modified Eagle’s minimum essential medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum, 5 mg/ml bovine insulin, and 0.2% glucose. To assess the expression of pro-inflammatory cytokines, confluent astrocytes were treated with 1 μg/ml LPS and/or HM. H₂ treatment was performed by replacing the astroglial medium with HM, and dehydrogenated HM was used as a control medium. The time course of H₂ concentration in HM was described previously^{12 (link)}. Replacement of the astroglial medium with HM or control medium was performed 3 h before the addition of LPS.