title: 4_3.7_D8
organism: Mus musculus
description: Protocols : Growth Protocol: mESC growth: Mouse embryonic stem cells (129B-13) were cultured in ESC media containing Knockout-DMEM (Thermo Fisher) with 15% EmbryoMax FBS (Millipore) and 20 ng/ml leukemia inhibitory factor (LIF, produced by Protein Expression Facility at EMBL Heidelberg), 1% non-essential amino acids, 1% Glutamax, 1% Pen/Strep, 1% of 55mM beta-Mercaptoethanol solution. Cells were maintained at 37C with 5% CO2. mESCs were used as neuronal differentiation Day 0 (D0). Neuronal Differentiation: mESCs were differentiated into glutamatergic neurons according to Bibel et al. (Nature Protocols, 2007) with small modifications. mESCs were resuspended in differentiation media without LIF and grown in suspension using non-adherent plates to promote the formation of embryoid bodies. Differentiation media was changed every two days. On day 4 retinoic acid was added to the media. Embryoid bodies were cultured for 4 additional days in the presence of retinoic acid to obtain neural precursor cells on differentiation day 8. For neural monolayer culture, plates were pre-coated with Poly-D-Lysin and Laminin. On day 8, Embryoid bodies were dissociated with trypsin and cells were plated in N2 media (regular DMEM supplemented with 1xN2 and 1xB27-VitaminA (Thermo Fisher)) at a density of 200.000 cells/cm2 on precoated plates. N2 media was changed every two days without exposing neuron culture to oxygen. Four days after plating, neurons were harvested after a total of 12 differentiation days (D12).
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2018-10-23T17:03:45Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2018-09-04T15:13:54Z
attributes.tag: External Id
attributes.value: SAMEA4883211
attributes.tag: INSDC center name
attributes.value: EUROPEAN MOLECUALR BIOLOGY LABORATORY, GENOME BIOLOGY UNIT
attributes.tag: INSDC first public
attributes.value: 2018-10-23T17:03:45Z
attributes.tag: INSDC last update
attributes.value: 2018-09-04T15:13:54Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: IndividualGeneticCharacteristics
attributes.value: Wild Type - CRISPR Control
attributes.tag: Submitter Id
attributes.value: embase-sample-21602:4_3.7_D8
attributes.tag: cell line
attributes.value: ESC 129-B13
attributes.tag: cell type
attributes.value: Neuronal Precursor Cells
attributes.tag: common name
attributes.value: house mouse
attributes.tag: developmental stage
attributes.value: Day eight
attributes.tag: sample name
attributes.value: embase-sample-21602:4_3.7_D8
attributes.tag: sampletype
attributes.value: frozen_sample
attributes.tag: scientific_name
attributes.value: Mus musculus
attributes.tag: sex
attributes.value: male