Catalog no 25030081

Human chordoma U-CH1, MUG-Chor1 and JHC7 cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). U-CH1 and MUG-Chor1 cells were cultured in RPMI-1640 medium (Catalog No. 11875093, Gibco, US) supplemented with an additional 1% l-glutamine (Catalog No. 25030081, Gibco, US), 10% characterized fetal bovine serum (FBS) (Catalog No. 10099141, Gibco, US), 10 units/mL penicillin and 10 mg/mL streptomycin (Catalog No. 10378016, Gibco, US). JHC7 cells were cultured in DMEM: F12 medium (ATCC^® 30-2006™, ATCC, USA) supplemented with 10% FBS, 10 units/mL penicillin and 10 mg/mL streptomycin. To culture U-CH1 and MUG-Chor1 cells, coating buffer (50 μg/mL rat tail type I collagen (Catalog No. 354236, BD Biosciences) was added to the culture flask for 1 h at room temperature prior to adding the cells. The SGC-7901 (a gastric cancer cell line)-shN and sh393 cells and GES-1 (an immortalized gastric epithelial cell line)-shN and sh393 cells were obtained as previously described [32 (link)]. All cells were maintained in humidified incubators at 37 °C with 5% CO₂.

The SaOS2-LM7 and its parent cell line SaOS2 were a kind gift from Dr. Eugenie Kleinerman, The University of Texas M.D. Anderson Cancer Center, USA. HOS (Catalog no. CRL-1543) and HOS-143B (Catalog no. CRL-8303) were purchased from ATCC. All cell lines were cultured in Minimal Essential Medium (MEM) (Catalog no. 10320-021, Gibco), supplemented with 10% fetal bovine serum (FBS) (Catalog no. 12483-020, Gibco), 1% penicillin-streptomycin (Catalog no. 15140-122, Gibco), 1mM sodium pyruvate (Catalog no. 11360-070, Gibco) and 2mM L-Glutamine (Catalog no. 25030-081, Gibco) at 37°C and 95% O2 and 5% CO2.

To isolate RGCs, we used a method that has previously been verified with slight modifications. Briefly, animals from each group were euthanized with CO₂ asphyxiation and their retinas were harvested in cold neurobasal medium and placed in a 37°C water bath for 5 minutes. The neurobasal media was removed and replaced with fresh pre-warmed neurobasal media containing papain (0.06mg/ml or 33.4 U/mg) and 5mM L-cysteine and incubated 20 minutes at 37°C. The papain solution was removed and replaced with neurobasal containing 2mM L-Glutamine (Gibco; Catalog no. 25030081) B-27 supplement (Gibco; Catalog no. 17504044) and 10% FBS (Gibco; Catalog no. 26140079). The retinas were dissociated by gently pipetting up and down with a wide bore 1ml pipette tip. Cells were then centrifuged at 450g for 8 minutes and resuspended in 90 μl of isolation buffer (DPBS + 0.5% BSA + 2mM EDTA) containing 25 μg/ml DNAse I + 5mM MgCl2. Cells were filtered through a 30μm cell strainer and incubated with CD90.2 magnetic beads (Miltenyi Biotec; Catalog no. 130-121-278) at 4°C for 10 minutes and then isolated using MACs LS (Miltenyi Biotec; Catalog no. 130-042-401) columns following the manufacturer’s instructions. After isolation, cells were washed with DPBS, centrifuged at 450g for 8 minutes and their pellets stored at −70°C prior to proteomics sample preparation and LC/MS analysis.