Homo sapiens
Protocols: Monolayer cells were maintained in T75 (10ml) or T175 (20ml) culture flasks, with the following media per flask: 500ml DMEM/F12 (Hyclone), 5% horse serum (Sigma), 0.5ug/ml hydrocortisone (Sigma, stock 1mg/ml in ethanol), 100ng/ul cholera toxin (Sigma, stock 1mg/ml in water), 10ug/ml insulin (Sigma) and 5ml Penicillin/Streptomycin (Pen/Strep) (Gibco, Invitrogen). The media mixture was finally sterilised using a 0.2um filter (Stericup, Millipore). 20ng/ml epidermal growth factor (Sigma) was added freshly to growth media after each cell splitting procedure. For the work performed in MCF10A 3D culture, cells were cultured on a laminin-rich extracellular matrix (ECM) Matrigel (Corning) to form acini. Cold Matrigel was applied to dishes or slides with a sterile pipette and spread evenly using an inoculation loop. Dishes or slides were then placed at 37C for at least 30 minutes to allow the matrix solidify. Prior to seeding of MCF10A cells onto laminin-coated tissue culture plastic/glass slides, cells were treated with Trypsin and detached from plastic flasks as described above, then resuspended in assay medium (containing 2% horse serum). In order to perform cell counting, an aliquot of cells was taken and mixed at a 1:1 ratio with Trypan Blue Stain (Invitrogen). Cells were then counted using a Countess Automated Cell Counter (Invitrogen). For RNA or protein extraction, cells were counted and seeded at a density of 200,000cells/6cm plastic dish (Greiner) pre-coated with 150ul of Matrigel.For all assays performed in 3D, medium was supplemented with 2% Matrigel and 5ng/ml EGF. RNA was harvested from dishes using the RNeasy Kit (Qiagen) or TRI Reagent (Sigma) according to manufacturers protocol. DNA was digested with the DNA-free Kit (Applied Biosystems). RNA concentrations were measured using a Nanodrop2000 (Thermo Scientific) and RNA quality was verified on a 2100 Bioanalyser (Agilent) using a Eukaryote Total RNA Nano Chip (version 2.6). Sequencing libraries were generated from 2ug of total RNA per sample with TruSeq RNA sample preparation Kit v2 (Illumina) according to manufacturers protocol. Size and purity of the libraries were analysed on a Bioanalyser High Sensitivity DNA chip (Agilent).
ENA-FIRST-PUBLIC
2014-10-02T17:04:17Z
ENA-LAST-UPDATE
2018-03-08T21:35:36Z
External Id
SAMEA2785581
INSDC center name
Systems Biology Ireland School of Medicine and Medical Sciences
INSDC first public
2014-10-02T17:04:17Z
INSDC last update
2018-03-08T21:35:36Z
INSDC status
public
Submitter Id
E-MTAB-2969:24h-2
broker name
ArrayExpress
cell line
MCF 10A
common name
human
growth condition
3D in matrigel for 24h
sample name
E-MTAB-2969:24h-2
scientific_name
Homo sapiens