E14 Tg2a embryonic stem cells (ESCs) were grown in Glasgow MEM (G-MEM; Gibco, Dublin, Ireland) supplemented with 15% FBS (Gibco, batch tested), 1% non-essential amino acids (NAA; Gibco), 1% sodium pyruvate (Gibco), 0.1% β-mercaptoethanol (Gibco), and 1000 units/mL LIF (ESGRO, Merck, Watford, UK). Differentiation of ESCs into embryoid bodies (EBs) and further into neuronal cells was carried out using a 4–/4+ retinoic acid (RA) procedure as previously described [32 (link),33 (link)]. Briefly, ESCs were seeded into 15 cm bacterial culture dishes at 12.5 × 106 cells/dish in EB medium (same as ESC culture medium, but 10% FBS and no LIF). After 4 days, EB medium + 5 μM all-trans RA (Sigma-Aldrich, Merck, Watford, UK) was used. After 4 more days, EBs were trypsinised and seeded onto culture dishes coated with poly-DL-ornithine (Sigma) and laminin (Roche Pharmaceuticals, Basel, Switzerland) at 1–2 x 105 cells per cm2 in N2 medium (DMEM/F-12 + N2-supplement; Invitrogen). From the next day onwards, cells were cultured in a 1:1 mixture of N2 and neurobasal medium (supplemented with B-27; and L-Glutamin; Invitrogen, Carlsbad, CA, USA) for 5 more days.
Dmem f 12 n2 supplement
Manufactured by Thermo Fisher Scientific
Sourced in Switzerland
DMEM/F-12 + N2-supplement is a cell culture medium formulation. It is designed to support the growth and maintenance of various cell types in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Sodium pyruvate, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
L glutamin, by Thermo Fisher Scientific (2 mentions)
Esgro, by Merck Group (2 mentions)
β mercaptoethanol, by Thermo Fisher Scientific (2 mentions)
Poly dl ornithine, by Merck Group (2 mentions)
Laminin, by Roche (2 mentions)
Poly l ornithine, by Merck Group (1 mentions)
Stempro accutase cell dissociation reagent, by Thermo Fisher Scientific (1 mentions)
Sb431542, by Merck Group (1 mentions)
Fibroblast growth factor 2 (fgf2), by Miltenyi Biotec (1 mentions)
Taqman assay, by Thermo Fisher Scientific (1 mentions)
Fibroblast growth factor 2 (fgf2), by Thermo Fisher Scientific (1 mentions)
Activin a, by Thermo Fisher Scientific (1 mentions)
Igf 1, by Merck Group (1 mentions)
Plo laminin, by Merck Group (1 mentions)
Laminin 521, by BioLamina (1 mentions)
Cedex hires analyzer, by Roche (1 mentions)
4 protocols using dmem f 12 n2 supplement
1
Differentiation of ESCs into Neuronal Cells
2
Differentiation of Embryonic Stem Cells into Neurons
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E14 Tg2a embryonic stem cells (ESCs) were grown in Glasgow MEM (G-MEM; Gibco, Dublin, Ireland) supplemented with 15% FBS (Gibco, batch tested), 1% non-essential amino acids (NAA; Gibco), 1% sodium pyruvate (Gibco), 0.1% β-mercaptoethanol (Gibco), and 1000 units/mL LIF (ESGRO, Merck, Watford, UK). Differentiation of ESCs into embryoid bodies (EBs) and further into neuronal cells was carried out using a 4−/4+ retinoic acid (RA) procedure as previously described [32 (link),33 (link)]. Briefly, ESCs were seeded into 15 cm bacterial culture dishes at 12.5 × 106 cells/dish in EB medium (same as ESC culture medium, but 10% FBS and no LIF). After 4 days, EB medium + 5 µM all-trans RA (Sigma-Aldrich, Merck, Watford, UK) was used. After 4 more days, EBs were trypsinised and seeded onto culture dishes coated with poly-DL-ornithine (Sigma) and laminin (Roche Pharmaceuticals, Basel, Switzerland) at 1–2 × 105 cells per cm2 in N2 medium (DMEM/F-12 + N2-supplement; Invitrogen). From the next day onwards, cells were cultured in a 1:1 mixture of N2 and neurobasal medium (supplemented with B-27; and L-Glutamin; Invitrogen, Carlsbad, CA, USA) for 5 more days.
3
Directed Differentiation of hESCs to NPCs
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To differentiate hESCs to neuronal progenitor cells (NPCs), H9-hESCs were initially cultured for 2 days in N2 media (DMEM/F12 + N2 supplement (Invitrogen)) containing 10 μm SB-431542 (Sigma) and 1 μm dorsomorphin (Calbiochem). After 2 days, hESCs were collected from plates using a cell-scraper. To generate embryoid bodies (EBs), hESCs were seeded in low-attachment plates using N2 media containing 10 μm SB-431542 (Sigma) and 1 μm dorsomorphin (Calbiochem). EBs were cultured for 4-6 days, with daily changes of media. Next, EBs were plated in Matrigel-coated plates (60-mm plates) and cultured during 5-7 days on NB medium (0.5x N2, Invitrogen; 0.5x B-27, Invitrogen; 20 ng/ml FGF-2, Miltenyi Biotec and 1% penicillin-streptomycin), replacing media every other day. After 5-7 days, neuronal rosettes were manually collected, dissociated and plated on poly-L-ornithine (Sigma)/laminin (Invitrogen) coated plates using NB medium. Upon reaching confluence, NPCs were detached from plates using StemPro Accutase Cell Dissociation Reagent (Invitrogen), passaging in a 1:3 ratio. NPCs were grown for < 10 passages.
4
Astrocyte Differentiation from ltNES Cells
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ltNES cells were plated at 60,000 cells/cm2 on 2 μg/cm2 poly-L-ornithine and 0.2 μg/cm2 laminin (PLO-Laminin) (Sigma, St. Louis, MO) double-coated culture vessels in FHIA-differentiation medium; DMEM/F12, N2 supplement (1:100; Invitrogen, Carlsbad, CA), B27 (1:100; Invitrogen), FGF2 (8 ng/mL; PeproTech, Rocky Hill, NJ), heregulin 1β (10 ng/mL; Sigma), IGF1 (200 ng/mL; Sigma), activin A (10 ng/mL; PeproTech). The medium was changed every other day and cells were passaged once they reached 80% confluence; 7–9 passages during the differentiation protocol of 28 days. This was performed for ltNES lines AF22, C1, and C9 generating the NES-Astro phenotype for each line. For a complete animal free system the regular B27 (0080085SA, 1:100, Invitrogen) was exchanged for Xenofree B27 (A1486701; 1:100; Invitrogen) and the double coating of PLO-Laminin (both from Sigma) for Laminin 521, 1 μg/cm2 (Biolamina, Sweden). Gene expression was assessed by applying real-time qPCR using TaqMan Assays (Applied Biosystems, Foster City, CA). Proliferation during differentiation was assessed by estimation of doubling time via cell count using a Cedex HiRes Analyzer (Roche, Switzerland).
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