Eagle s minimum essential medium

KPL-1 human breast cancer cells, which were established from the carcinomatous effusion of a postmenopausal patient with breast cancer who had become resistant to hormone therapy (22 (link)), were purchased from Kawasaki Medical School (Okayama, Japan) and maintained in Dulbecco’s modified Eagle’s medium (Wako, Osaka, Japan) supplemented with 10% fetal bovine serum (BioWest, Logan, UT, USA) and 1% penicillin-streptomycin solution (Cosmo Bio Company, Tokyo, Japan). BALB-MC mouse breast cancer cells, which were established from a spontaneous mammary tumor in a 17-month-old female BALB/c mouse (23 (link)), were purchased from the Japanese Collection of Research BioResources (Osaka, Japan) and maintained in Eagle’s minimum essential medium (Wako) supplemented with 10% fetal bovine serum (BioWest) and 1% penicillin-streptomycin solution (Cosmo Bio Company).

Human neuroblastoma, SH-SY5Y cells, maintained in a 1:1 mixture of Eagle’s minimum essential medium (Wako) and Ham’s F12 medium (Wako) containing 10% fetal bovine serum (Biological Industries), were used as a neuronal cell model to estimate the neurotoxicity of each Aβ with a slight modification to the previously described method24 (link). In brief, each Aβ was dissolved in 0.1% NH₄OH to generate a 10X stock solution. The resultant peptide solution (10 μL) was diluted with 0.1% NH₄OH to appropriate final concentrations in medium before being added to 100 μL of the culture medium of near-confluent cells (10⁴ cells/well) after one or two overnight incubation. In the case to test the effect of antibodies on the cells, the culture medium was replaced with fresh medium containing pre-incubated (30 min) Aβ solution with antibodies. After being treated at 37 °C for 16 or 48 h, 10 μL of 5 mg/mL MTT (Sigma) was added to cells, followed by incubation for 4 h at 37 °C. After removing the medium, 100 μL cell lysis buffer (10% SDS, 0.01 M NH₄Cl) was subsequently added to the cells. The resulting cell lysate was subsequently incubated overnight in the dark at room temperature before absorbance measurements were made at 595 nm with a microplate reader (MultiScan JX; Thermo Scientific). Absorbance obtained by the addition of vehicle (0.1% NH₄OH) was taken as 100%.

Prostate PC3 and renal A498 cancer cell lines were obtained from the NCI-60 panel (National Cancer Institute, Bethesda, MD, USA). Bladder J82 and lung A549 cancer cell lines were purchased from the American Type Culture Collection (ATCC) (Rockville, MD, USA). NIH3T3 mouse embryonic fibroblasts were purchased from ATCC, and GGCT was overexpressed using the pCX4bsr retroviral vector as previously described^{2 (link)}. PC3, J82, and NIH3T3 cells were cultured in Dulbecco’s modified Eagle’s medium (#043-30085, Wako Pure Chemical Industries, Osaka, Japan), A498 cells in Eagle’s minimum essential medium (#055-08975, Wako), and A549 cells in RPMI-1640 (#05918, Nissui, Tokyo, Japan) supplemented with 300 μg/mL _L-glutamine and 1 mmol/L sodium pyruvate. All growth media were supplemented with 10% FBS (#104037, Gibco, Waltham, MA, USA), 50 units/mL penicillin, and 100 μg/mL streptomycin. All cells were maintained at 37 °C in a 5% CO₂ atmosphere.

LS180 and HCT116 (colon cancer) cells were obtained from DS Pharma Biomedical (Osaka, Japan) and Dr. B. Vogelstein (Johns Hopkins University, Baltimore, MD), respectively.
LS180 cells were cultured in Eagle's Minimum Essential Medium (Wako, Osaka, Japan). HCT116 cells were maintained in Dulbecco's Modified Eagle's Medium (Wako). All culture mediums were supplemented with 10% (v/v) heat-inactivated fetal bovine serum and antibiotics. The cells were grown at 37°C with 5% CO₂.