Mt10013cv

Primary astrocytes were prepared from the cortex of P2 CD1 mice. Cortices were dissected in ice-cold DPBS and digested in 20 U/mL papain for approximately 10 minutes at 37°C, washed in neuronal medium and strained to remove cellular debris. Cells were plated in T25 flasks in glial medium (DMEM (FisherScientific, Cat#MT10013CV), 10% fetal bovine serum (Atlanta Biologicals Cat#S10350), 25 U/mL penicillin/streptomycin (ThermoFisher 15140122)). Cells were placed in the incubator at 37°C for 2 hours. After 2 hours, cells were washed twice with 4°C MEM (Sigma-Aldrich Cat#M2279) and media was replaced with 4°C glial medium. When flasks reached confluence, they were shaken roughly to remove all remaining non-astrocytic cells. For astrocyte and hippocampal neuron co-culture, astrocytes were plated on 96-well plates that had been coated with 40 μg/mL poly-D-lysine (PDL, Sigma-Aldrich Cat#P0899) and 1.15 mg/mL laminin (EMD Millipore Cat#CC095). Astrocytes were lifted from the flask with trypsin and plated in 96-well plates for imaging (8,500 cell/well) or 6-well plates for biochemistry (500,000 cells/well). When cells reached 70% confluence 5-fluorodeoxyuridine (5-FDU) solution (6.7 mg/mL 5-FDU (Sigma-Aldrich Cat#F0503), 16.5 mg/mL uridine (Sigma-Aldrich Cat#U3003)) was added to slow cell division.

MEFs and mouse breast tumor lines: DMEM (Corning mt10013cv) supplemented with 10% FBS (Atlanta Biologicals), 1% pen/strep (Fisher 30002ci) and 2mM L-glutamine (total 6mM) (Fisher MT25005CI). MDA-MB468, MDA-MB 231, Myc-CaP (2015), and U87: DMEM supplemented with 10% FBS and 1% pen/strep. HCC1419, HCC1187, HCC 1937, HCC 1806, BT549, ZR75-1, PC3, LNCAP, DBTRG: RPMI (Fisher 10040cv) supplemented with 10% FBS and 1% pen/strep. CaP8 cells (2015): DMEM with 10% FBS, 1% pen/strep, and 5ug/mL insulin (Sigma I9278). Neurospheres: stem cell media with 10ug/mL FGF (R&D Systems 233-FB-025), 20ug/mL EGF (Peprotech AF-100-15) and heparin (from Dr. Raymund Yong, May 2015). All cells were cultured in a 37°C incubator with humidity and 5% CO₂. Cell lines were obtained from ATCC (which authenticate cell lines using several methods including DNA fingerprinting) in 2006, with the exception of MEFs, MCCL-278, and MCCL-357 which were produced in our lab from mice (2012-2016). Cell lines were clear of mycoplasma as determined by the Lonza kit (LT07-418) within 6 months of their use. Cell lines were further authenticated in 2015 by LabCorp using a short tandem repeat method.