Dmem f 12 l glutamine

LIM1215, HCA7 and HEK-293T cell lines were cultured in DMEM/F-12 + L-Glutamine + 15 mM HEPES (Gibco) supplemented with 10% fetal bovine serum (FBS) (HyClone) and 1% penicillin/streptomycin (Gibco) at 37°C with 5% CO₂. LIM1215 cells were obtained from the Ludwig Institute for Cancer Research, New York, NY, USA and HCA7 and HEK-293T cells were obtained from the American Type Culture Collection (ATCC).
Cetuximab (Erbitux^®) was purchased from Merck Serono, lapatinib (Tykerb/Tyverb^®) from GlaxoSmithKline, and trastuzumab (Herceptin^®) and pertuzumab (Perjeta^®) from Roche. Gefitinib (ZD1839), GDC-0941, trametinib (GSK1120212) and afatinib (BIBW2992) were purchased from Selleck Chemicals. NRG1 was purchased from Peprotech.

Mixed glial cultures were obtained from the cortices of P0–P2 wt and bid^−/− mice of mixed sexes on a C57BL/6 background, as described previously [51 ]. Briefly, the cortices were isolated, the meninges were removed, and the tissue was incubated with Trypsin-EDTA at 37 °C for 10 min. The Trypsin-EDTA was removed and replaced with DMEM-F12/L-glutamine (Gibco, Life Technologies) containing Penicillin-Streptomycin (1%, Sigma-Aldrich) and fetal bovine serum (10%, Sigma-Aldrich). The cells were triturated and passed through a 40-μm nylon cell strainer (BD Falcon) before being centrifuged at 300 × g for 5 min. The cells were plated at a density of 2 cortices/T75 flask and cultured for 10 days in the presence of M-CSF (10 ng/ml, R & D Systems) and GM-CSF (20 ng/ml, R & D Systems) in order to promote microglial proliferation [52 (link)]. Microglia were isolated from the co-culture, and the remaining cells were passaged twice and cultured in the absence of M-CSF and GM-CSF as astrocyte cultures. Bid^−/− mice were generated in the laboratory of Dr. Andreas Strasser, WEHI, Melbourne, Australia [53 (link)].