Epidermal growth factor egf

The human neural stem cell line ReNcell VM was kindly provided by Dr. A. Cuadrado [24 (link)]. ReNcell VM cells were cultured on Corning Matrigel hESC-Qualified Matrix (Corning, Corning, NY, USA)-coated plates with proliferation medium: neurobasal medium (Gibco, Waltham, MA, USA) supplemented with 2% (v/v) B27 supplement (Gibco), 2 mM glutamax (Gibco), 50 μg/mL gentamicin, 20 ng/mL b-FGF (Basic Fibroblast Growth Factor; Peprotech, Waltham, MA, USA), and 20 ng/mL EGF (Epidermal Growth Factor; Peprotech), as described in [24 (link)]. Cells were cultured at 37 °C in a 5% CO₂ atmosphere, and cell passages were performed every 3–4 days.
At 80–90% confluence, differentiation was induced by growth factor withdrawal from the proliferation medium and monitored using phase-contrast microscopy and expression analysis of neuronal precursor (Nestin, SOX-2, and Ki-67 (MKI67)), neuronal (β-tubulin III, synapsin I (SYN1), and synaptophysin (SYP)), dopaminergic (tyrosine hydroxylase (TH) and dopamine decarboxylase (AADC)), and glial markers (glial fibrillary acidic protein (GFAP) and oligodendrocyte transcription factor (OLIG2)). Meanwhile, the differentiation medium was changed every 2–3 days.

Six- to eight-week old C57BL/6 female mice were injected with LV PGK-GFP in the DG. Three months later, hippocampi were isolated from these mice and used to prepare hippocampal progenitor cells as previously described (Ray and Gage, 2006 (link); Suh et al., 2007 (link)). Briefly, 3–5 LV PGK-GFP injected hippocampi were isolated from these mice and digested in PPD solution [papain (2.5 U/ml, Worthington), pronage (1 U/ml, Roche), and DNase (250 U/ml, Worthington)] and sucrose gradient was applied to remove cell debris and myelin. Then cells were plated in the presence of FGF2 (Fibroblast Growth Factor, 20 ng/ml; PeproTech), EGF (Epidermal Growth Factor, 20 ng/ml; PeproTech), and heparin (5 μg/ml; Sigma) in DMEM/F12 (Life Technology) basal media supplemented with N2 (Life Technology). Once NSCs were established and expanded in the presence of FGF2 and EGF, we sorted out GFP-expressing cells via FACS and established three clonally expanded NSCs lines. The number of total GFP-expressing cells isolated by FACS was around 2 × 10ˆ4 cells per preparation. To differentiate GFP-expressing NSCs, 10⁵ cells/cm² were plated on the laminin-coated glass chamber slides (Nalge Nunc International) and cultured in differentiation medium consisting of DMEM/F12, N2 supplement, and 5 μM forskolin (Sigma), for 7 more days.

Three different breast cell lines, including non-malignant (MCF10A), malignant (MCF7), and metastatic (MDA-MB-231) cells were grown. MCF10A were cultured in DMEM/Ham’s F-12 (Sigma-Aldrich, Milano, Italy) supplemented with 100 ng/mL cholera toxin, 20 ng/mL EGF epidermal growth factor, 0.01 mg/mL insulin, 500 ng/mL hydrocortisone, and 5% horse serum (Life Technologies, Monza, Italy). MCF7 and MDA-MB-231 cells were cultured in the DMEM medium (DMEM, Life Technologies, Monza, Italy) and supplemented with 2 mM L-glutamine, (Sigma-Aldrich, St. Louis, MO, USA), 10% heat-inactivated fetal bovine serum (FBS) (Thermo Scientific), 1% penicillin/streptomycin (Life Technologies, Monza, Italy) and 0.25 ug/mL amphotericin B.
Cells were grown on poly-lysine coated glass microscopy slides (Fisher Scientific, Rodano, Italy). The slides were located at the bottom of petri dishes incubated at 37 °C, and 5% CO₂. Before FTIR measurements, the cells were fixed by means of 3.7% PFA in PBS solution and preserved inside a desiccator.

We isolated primary NSCs from the ventricular zone of single wild type mouse brains (C57BL/6J, Harlan, The Netherlands) at embryonic day 14 (E14) as described before [21 (link),22 (link)]. Primary cultures of neurospheres (NSPs) were kept under proliferating conditions in neurobasal medium (DMEM F12; Lonza, Basel, Switzerland) supplemented with 1% B27 without vitamin A (Life Technologies, Carlsbad, CA, USA), penicillin (100 U/mL; Lonza), streptomycin (100 g/mL; Lonza), and 20 ng/mL EGF (Epidermal Growth Factor; Life Technologies). We differentiated NSPs into astrocytes on 6-well or 12-well plates coated with poly-L-ornithine and by exchanging the proliferation medium with DMEM containing 10% FBS (Fetal Bovine Serum; Gibco, Grand Island, NE, USA), penicillin (100 U/mL; Lonza), and streptomycin (100 g/mL; Lonza). We differentiated cells into astrocytes at different time points (24, 48, 72 h and 1 week) at 37 °C in 5% CO₂/95% air atmosphere, and we kept them under normal conditions or treated with TNF (50 ng/mL; R&D Systems, Minneapolis, MN, USA).