Dulbecco s modified eagle

Cells were cultured in an incubator at 37 °C, with 5% (v/v) CO₂ atmosphere and 95% relative humidity. The human hepatocellular carcinoma (HCC) cell line HuH7 (JCRB0403; Japanese Collection of Research Bioresources Cell Bank, Osaka, Japan) was grown in Dulbecco's Modified Eagle Medium (1 g/l glucose; Sigma Aldrich, St. Louis, Missouri, USA) supplemented with 10% (v/v) fetal bovine serum (FBS; FBS Superior, Biochrom GmbH, Berlin, Germany) and 100 U/ml penicillin and 100 µg/ml streptomycin (P/S; Sigma-Aldrich). The human MSC line (HSP70B-NIS- MSC) was cultured in Roswell Park Memorial Institute (RPMI)-1640 culture medium (Sigma- Aldrich) enriched with 10% FBS, P/S and G-418.

In a series of experiments each aneurysm tissue sections were incubated in medium in the presence and absence of DL-homocysteine. Aneurysm fragments were cut into approximately 2 mm pieces. Equal wet weights of the tissue were placed in each well of 12-well plates with serum-free Dulbecco's Modified Eagle's Medium (DMEM) with 4500 mg/L glucose (without phenol red and L-glutamine; Sigma-Aldrich, St. Louis, Missouri, USA). The thick and thin ILTs and adjacent walls were separately incubated without or with 100 μmol/L or 500 μmol/L DL-homocysteine (DL-homocysteine ≥ 95%, Sigma-Aldrich) at 37°C for 6 h in humidified air with 5% v/v CO₂. From the wide range of DL-Hcy concentrations mentioned in the literature (10-2000 μmol/L) the two values were selected (as discussed). The incubation times were chosen based on previous studies [29 (link)]. Nontreated aneurysm tissues were used in each experiment as controls. After treatment, tissue samples were collected for protein extraction.

The T2 cell line (HLA A2.1⁺) was maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) (Sigma Aldrich, St Louis, MO), 2 mM L-glutamine, 10 mM HEPES, 100 U ml⁻¹ penicillin, 100 μg ml⁻¹ streptomycin.
The Huh-7-Lunet cell line designated Lunet-HLA-A2-luc/neoET (Huh7^A2HCV^Rep) has been described previously30 (link). The Lunet-HLA-A2-luc/neoET has ectopic HLA-A2 expression and contains a selectable HCV sub-genomic RNA replicon of genotype 1b (Con1-ET) that also expresses the firefly luciferase gene fused to the selectable marker by ubiquitin. The Lunet-HLA-A2-luc/neoET cell line was maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS) (Sigma Aldrich, St Louis, MO), 2 mM L-glutamine, 100 U ml⁻¹ penicillin, 100 μg ml⁻¹ streptomycin, 1 mM nonessential amino acids, and puromycin (1 μg/ml) and G418 (0.5 mg/ml) (Sigma Aldrich). All cells were grown in a humidified 37 °C and 5% CO₂ incubator.

Neuro2a and HeLa cells were obtained from JCRB, and maintained in Dulbecco’s modified Eagle’s medium (DMEM, Sigma) supplemented with 10% FBS (Invitrogen). Caenorhabditis elegans strain was derived from the wild type Bristol strain N2 and was maintained by standard techniques [20 ]. Anesthetics used in this study; isoflurane (Escain, Pfizer), pentobarbital sodium (Somnopentyl, Kyoristu Seiyaku), 1-phenoxy-2-propanol (Wako), Lidocaine (xylestesine-A, GmbH).